Compositions and methods for treating retinal degradation

ABSTRACT

The present disclosure relates to compounds, compositions, and methods useful for treating retinal damage and/or retinal degradation/retinal degeneration, for inhibiting inflammasome activation by Alu RNA associated with a cell, for reducing ATP-induced permeability of a cell, for reducing an amount of mitochondrial reactive oxygen species in a cell, and for reducing an amount of mitochondrial reactive oxygen species in a cell. The present disclosure further relates to compounds, compositions, and methods for use in protecting an RPE cell and/or for treating, including prophylactic and therapeutic treatment, of conditions associated with retinal damage and/or degradation including, but not limited to, dry age related macular degeneration (AMD) and wet AMD, Alzheimer disease, various forms of arthritis, atherosclerosis, diabetes mellitus, chronic obstructive pulmonary disease, inflammatory bowel disease, and Duchenne muscular dystrophy.

RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. patentapplication Ser. No. 15/552,441, now-allowed, International PatentApplication No. PCT/US2016/019852 filed Feb. 26, 2016, and U.S.Provisional Patent Application Nos. 62/247,099, filed Oct. 27, 2015;62/246,455, filed Oct. 26, 2015; and 62/121,379, filed Feb. 26, 2015,the entire disclosures of which are incorporated herein by thisreference.

TECHNICAL FIELD

The present disclosure relates to compounds, compositions, and methodsuseful for treating retinal damage and/or retinal degradation/retinaldegeneration, for inhibiting inflammasome activation by Alu RNAassociated with a cell, for reducing ATP-induced permeability of a cell,for reducing an amount of mitochondrial reactive oxygen species in acell, and for reducing an amount of mitochondrial reactive oxygenspecies in a cell. The present disclosure relates to compounds andcompositions comprising a nucleoside and/or a nucleoside reversetranscriptase inhibitor (NRTI).

BACKGROUND

Geographic atrophy, an advanced form of age-related macular degenerationthat causes blindness in millions of people worldwide and for whichthere is no approved treatment, results from death of retinal pigmentedepithelium (RPE) cells. For example, expression of DICER, an enzymeinvolved in microRNA (miRNA) biogenesis, is reduced in the RPE of humaneyes with geographic atrophy, and that conditional ablation of Dicer1induces RPE degeneration in mice. Surprisingly, ablation of seven otherenzymes responsible for miRNA biogenesis or function does not inducesuch pathology. Instead, knockdown of DICER1 leads to accumulation ofAlu repeat RNA in human RPE cells and of B1 and B2 (Alu-like elements)repeat RNAs in the RPE of mice. Alu RNA is dramatically increased in theRPE of human eyes with geographic atrophy, and introduction of thispathological RNA induces death of human RPE cells and RPE degenerationin mice.

Age-related macular degeneration (AMD), which is as prevalent as cancerin industrialized countries, is a leading cause of blindness worldwide.In contrast to the neovascular form of AMD, for which many approvedtreatments exist, the far more common atrophic form of AMD remainspoorly understood and without effective clinical intervention. Extensiveatrophy of the retinal pigment epithelium leads to severe vision lossand is termed geographic atrophy.

Hence, there remains a need for compositions and methods for treatingretinal degradation/retinal degeneration, and particularly RPEdegradation.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are used, and the accompanyingdrawings of which:

FIG. 1 displays a top row of ocular fundus photographs of mice receivingcontrol PBS, or Alu RNA treatment, with or without increasing amounts ofd4T (left to right); and RPE flat mounts, stained for intercellularjunctions (ZO-1) in red that are disrupted upon Alu RNA administrationbut that are restored to healthy RPE morphology/intercellular junctionsat highest dose of d4T.

FIG. 2 provides a bar graph showing that human (HeLa) cells treated withan enforced expression plasmid for Alu RNA (pAluA) for denoted amountsof time exhibited profoundly reduced viability compared to a nullplasmid (pUC19), as monitored by MTS proliferation assay and that d4Tco-administration prevented cell death induced by Alu overexpression.

FIG. 3 shows the results of Northern blotting using an Alu-specificprobe. As presented in FIG. 3, primary human RPE cells treated withantisense oligonucleotides targeting DICER1 (Dcr as) (lane 3 (third lanefrom left)) show increased Alu RNA levels in the nuclear compartmentcompared to control antisense oligonucleotides (Ctr as) (lane 1(leftmost)), and co-administration of d4T (lanes 2 and 4) does notreduce Alu RNA levels. u6 (bottom row) is shown as a loading control fornuclear fraction.

FIG. 4 provides another example of the results of Northern blottingusing an Alu-specific probe. As presented in FIG. 4, co-administrationof d4T does not change Alu RNA levels at 1, 4, or 24 hours aftertransfection in the nuclear fraction of human RPE cells transfected withAlu RNA, with or without d4T, as detected by Northern blotting using anAlu-specific probe. u6 (bottom row) is shown as loading control fornuclear fraction in FIG. 4.

FIG. 5 provides the results of a Western blot showing that Alu RNAcauses Caspase-1 maturation in primary human RPE cells at 24 hours afterAlu administration (top, middle lane, lower band), which is blocked byco-treatment with d4T (100 uM; rightmost lane). The bottom row is avinculin loading control.

FIG. 6 is a Western blot showing that Alu RNA causes Caspase-1maturation in primary human RPE cells at 24 hours after Aluadministration (top, middle lane, lower band), which is blocked withco-treatment with 3TC (20-100 uM; rightmost lane), wherein the lowermostband is the loading control, vinculin.

FIG. 7 is a Western blot showing that Alu RNA causes Caspase-1maturation in primary human RPE cells at 24 hours after Aluadministration (top, middle lane, lower band), which is blocked withco-treatment with azidothymidine (AZT), cordycepin, and abacavir (ABC)(50-100 uM; lanes 3-8 from left). The loading control vinculin is shownon the bottom.

FIG. 8 provides a gel showing that primary human RPE cells treated withLPS/ATP, a classic inflammasome activator, exhibit increased Casp-1activation, and phosphorylation of IRAK4, which is also a marker ofinflammasome signaling via the cell surface receptor adaptor proteinMyD88. Moreover, as shown in FIG. 8, d4T (25/100 uM) blocks Casp-1activation and IRAK4 phosphorylation induced by LPS/ATP. Vinculin wasused as the loading control in the gel of FIG. 8. Additionally, asshown, LPS and ATP activate the NLRP3 inflammasome only in combination.

FIG. 9 provides the results of Western blotting, wherein d4T, 3TC, andcordycepin (at 100 uM), all di-deoxy nucleoside reverse transcriptaseinhibitors, are shown to inhibit Caspase-1 activation (active p20 band,top) and IL-18 maturation (bottom) induced by LPS/ATP. To produce FIG.9, cell culture supernatants were collected after (i) no treatment, (ii)LPS treatment, or (iii) LPS/ATP treatment of mouse bone marrow-derivedmacrophages and run on Western blotting probing with antibodies forCaspase-1 and IL-18.

FIG. 10 provides the result of a Western blot showing that d4T (100, 250uM) inhibits IL-1 beta maturation (top, 18 and 22 kDa forms) andCaspase-1 activation (active p20 band, bottom) induced by nigericin. Toproduce FIG. 10, cell culture supernatants were collected after (i) notreatment, (ii) LPS treatment, or (iii) LPS/nigericin treatment of mousebone marrow-derived macrophages and run on Western blotting probing withantibodies for IL-1 beta and Caspase-1.

FIG. 11 shows a bar graph illustrating that d4T does not inhibit IL-1beta secretion from PMA-differentiated THP-1 monocytes induced bymonosodium urate (MSU). FIG. 11 was created after human THP-1 monocyteswere differentiated into macrophages with PMA, and, as shown in FIG. 11,treatment with MSU, a known inflammasome activator, increased IL-1 betasecretion compared to non-treated cells, whereas d4T co-administrationat a range of doses (25-1000 uM) did not significantly affect IL-1betasecretion.

FIG. 12 is a bar graph, which shows that d4T and other nucleosidereverse transcriptase inhibitors do not inhibit IL-1 beta secretion fromPMA-differentiated THP-1 monocytes induced by MSU. Human THP-1 monocyteswere differentiated into macrophages with PMA. Their treatment with MSUincreased IL-1 beta secretion compared to non-treated cells, as shown inFIG. 12, while co-administration of d4T, 3TC, or cordycepin (all aredi-deoxy nucleotide analogs) at a range of doses (25-1000 uM) did notsignificantly affect IL-1beta secretion.

FIG. 13 is a graph, which provides that d4T reduces NLRP3 priminginduced by Alu RNA. Indeed, as shown in FIG. 13, Alu RNA transfectionincreases NLRP3 mRNA levels in primary human RPE cells at 16 hours, anevent termed “priming” (Y-axis) compared to mock (transfection reagentalone). This effect is blunted by co-administration of d4T (100 uM) andnormalized to 18S RNA control.

FIG. 14 illustrates, in graph format, that Alu RNA transfectionincreases IL-1 beta mRNA levels in primary human RPE cells at 24 hours,an event termed “priming”, (Y-axis) compared to mock (transfectionreagent alone). This effect is blunted by co-administration of d4T (100uM) and normalized to 18S RNA control.

FIG. 15 shows that d4T reduces mitochondrial ROS caused by Aluexpression. Indeed, FIG. 15 demonstrates that enforced expression of Alu(pAluA) causes increased mitochondrial reactive oxygen species (mtROS),as detected by MitoSox assay. In order to produce FIG. 15, primary humanRPE cells were incubated with Alu expressing plasmid or control plasmid(pUC19) with or without d4T. After 15 hours cells were co-stained formtROS (red) and for cell count, nuclei (blue; Hoechst DNA stain). Cellsin the pAluA group exhibited greater mtROS staining (red) compared topUC19 control, an effect that is reduced in pAluA+d4T treated cells.

FIG. 16 provides a graph showing that d4T does not inhibit ATP releaseinduced by Alu RNA. Moreover, primary human RPE cells treated with AluRNA, for the times indicated in FIG. 16, release ATP. Cell culturesupernatant was collected from mock or Alu RNA treated cells, with orwithout d4T, and ATP was detected using an ATP-dependent luciferaseassay. Notably, d4T did not affect ATP release.

FIG. 17 shows that d4T reduces ATP-induced cell permeability to Yo-Pro1(P2X7 receptor assay). Indeed, d4T dose-dependently reduced Yo-Pro entryinduced by ATP, determined by an area-scan fluorescent measurement in a96 well microplate reader. FIG. 17 provides the results of thefluorescence measurement in relative fluorescence units (RFU, y-axis).

FIG. 18 illustrates, in graph format, that d4T reduces extracellularpotassium levels, which increase after Alu RNA transfection. Indeed,cell culture potassium levels increase in primary human RPE cellstreated with Alu RNA for 6 hours, an effect that is reduced by d4Tco-administration. Potassium levels were determined in cell culturesupernatants spectrophotometrically using a potassium-dependent pyruvatekinase assay.

FIG. 19 shows that d4T blocks bzATP-induced cell permeability to Yo-Pro1(P2X7 receptor assay). To prepare FIG. 19, d4T blocked YO-PRO-1 iodideentry in HEK293 cells stably expressing the human P2X7 receptorstimulated with the P2X7-selective agonist bzATP. Cells werepre-incubated with d4T for 30 minutes prior to addition of bzATP/YO-PRO,and fluorescence (in relative fluorescence units) at 485/515 nm wasmeasured at t=30 minutes.

FIG. 20 provides a chemical structure of methoxy-d4T (me-d4T; IUPACname:1-[(2R,5S)-5-(methoxymethyl)-2,5-dihydrofuran-2-yl]-5-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione,also referred to herein “Kamuvudine 1”). More specifically, as shown inFIG. 20, a single substitution of the ribose 5′ hydroxyl group of d4Twith a methoxy group (circled) has been designed to prevent d4Tphosphorylation.

FIG. 21 is a Western blot of Caspase-1 activation (p20 subunit) inprimary human RPE cells transfected with Alu RNA±me-d4T.

FIG. 22 shows cells, wherein unmodified d4T, but not me-d4T, blocksreplication of a GFP-expressing lentivirus in HeLa cells.

FIG. 23 provides a graph illustrating that unmodified d4T, but notme-d4T, reduces mtDNA levels (normalized to chromosomal DNA exon-intronjunction sequence) in primary mouse RPE cells as determined by real-timequantitative PCR. n=4, *p<0.05 by Student's t-test.

FIG. 24 provides flat mounts stained for zonula occludens-1 (ZO-1; red),bottom row. Degeneration outlined by blue arrowheads. Representativeimages of n=4 (B, C, E) shown. Scale bars, (C): 200 μm; (E): 20 μm.

FIG. 25 provides a schematic overview of me-d4T synthesis.

FIG. 26 is an HPLC chromatogram of me-d4T (peak #6) final product, >97%purity.

FIG. 27 is a 1H NMR spectroscopy of me-d4T final product, wherein thechemical shifts are consistent with the structure of me-d4T.

FIG. 28 provides the results of liquid chromatography/mass spectrometryof me-d4T final product, m/z ratio consistent with the structure ofme-d4T.

FIG. 29 provides the methoxy variant of a nucleoside analog. Thechemical structure of 3TC (2′,3′ dideoxycytidine) is shown, wherein themethoxy variation (O-methyl group) of nucleoside analog is circled.

FIG. 30 provides the methoxy variant of a nucleoside analog. Thechemical structure of AZT (3′-azido-2′,3′-dideoxythymidine) is shown,wherein the methoxy variation (O-methyl group) of nucleoside analog iscircled.

FIG. 31 provides the methoxy variant of a nucleoside analog. Thechemical structure of ABC (cyclopropylaminopurinylcyclopentene) isshown, wherein the methoxy variation (O-methyl group) of nucleosideanalog is circled.

FIG. 32 shows a cell permeant variant of d4T (IC-d4T), where “n” groupis equal to 11. Derivatives include cell permeant variants of 3TC, AZT,ABC, where the nucleobase group (circled) may be replaced, in variousembodiments, by 3TC, AZT, ABC, or methoxy-variants of d4T, 3TC, AZT, ABC(FIG. 29-31), or derivatives thereof.

FIG. 33 provides the structure of an exemplary NRTI according to thepresent disclosure.

FIG. 34 is a Western blot of Caspase-1 activation (p20 subunit) andIRAK4 phosphorylation in primary human RPE cells transfected with AluRNA±d4T.

FIG. 35 is a Western blot of Caspase-1 activation in human RPE cellstransfected with Alu RNA±NRTIs (3TC, AZT, ABC).

FIG. 36 includes fundus photographs: top row; flat mounts stained forzonula occludens-1 (ZO-1; red), bottom row. Scale bars, 50 μm.

FIG. 37 provides fundus photographs: top row; flat mounts stained forzonula occludens-1 (ZO-1; red), bottom row. Scale bars, 50 μm.

FIG. 38 illustrates that NRTIs block LPS/ATP-induced inflammasomeactivation. Specifically, FIG. 38 shows a gel indicating that d4Tblocked Caspase-1.

FIG. 39 also illustrates that NRTIs block LPS/ATP-induced inflammasomeactivation, showing specifically a gel indicating that d4T blocked IL-1beta.

FIG. 40 presents chromatograms showing that Raji TK⁺ cells, but not RajiTK⁻ cells, phosphorylate AZT to AZT-triphosphate (AZT-TP) as determinedby liquid chromatography-mass spectrometry (LC-MS).

FIG. 41 shows that AZT blocks IL-1 beta activation by LPS/ATP in bothRaji TK⁻ and TK⁺ cells, as determined by Western blot of cell lysates.

FIG. 42 is a bar graph illustrating that d4T does not block Alu-inducedATP release from primary human RPE cells (n=4).

FIG. 43 provides a graph of P2X7-mediated YO-PRO-1 dye uptake(fluorescence) induced by bzATP (100 μM) in HEK293 cells stablyexpressing the human P2X7 receptor was inhibited by d4T and A438079 (64μM for both drugs). Fluorescence values are baseline subtracted fromcells without bzATP treatment. * bzATP vs. d4T; #bzATP vs. A438079,p<0.05 by Student-Newman Keuls post-hoc test (n=12).

FIG. 44 is a Western blot of Caspase-1 activation (p20 subunit) andIRAK4 phosphorylation in primary mouse RPE cells transfected with AluRNA±d4T.

FIG. 45 is a Northern blot of biotin-UTP-labeled Alu RNA-transfectedprimary human RPE cells.

FIG. 46 provides LC-MS/MS spectra of AZT-triphosphate (AZT-TP).

FIG. 47 provides LC-MS/MS spectra of AZU-triphosphate (AZT-TP).

FIG. 48 shows the chromatographic separation of Raji TK⁻ cells spikedwith AZT-TP with MS spectra (inset) to confirm identity of designatedpeaks.

FIG. 49 shows the chromatographic separation of Raji TK⁻ cells spikedwith AZU-TP with MS spectra (inset) to confirm identity of designatedpeaks.

FIG. 50 is a standard curve of AZT-TP standards (black circle). Asshown, Raji TK⁺ samples treated with AZT produced AZT-TP (whitetriangles), whereas AZT-TP was not detectable in Raji TK⁻ cells treatedwith AZT.

FIG. 51 is a Western blot of Caspase-1 activation (p20 subunit) inprimary human RPE cells transfected with Alu RNA, with short peptide(Panx1¹⁰), which blocks P2X7 pore function but not cation flux (vs.scrambled peptide: Scr Panx1¹⁰).

FIG. 52 is a Western blot of Caspase-1 activation (p20 subunit) inprimary human RPE cells transfected with Alu RNA, with calmidazolium(FIG. 32 provides the chemical structure of IC- and EC-d4T used), whichblocks P2X7 cation flux but not pore function.

FIG. 53 is a Western blot of Caspase-1 activation (p20 subunit) inprimary human RPE cells transfected with Alu RNA, with cell permeable(IC), cell-impermeable (EC), or unmodified (no tag) d4T.

FIG. 54 shows that d4T prevents pAlu-induced mitochondrial ROSgeneration in primary human RPE cells. In FIG. 54, mitochondrialreactive oxygen species (ROS) were visualized with MitoSox (Red) andcell nuclei with Hoechst (Blue).

FIG. 55 is a western blot of Caspase-1 activation (p20 subunit) inprimary human RPE cells in the presence of iron (III) ammonium citrate.The addition of TM-3TC (Structure 8) (25 μM) blocked iron-inducedCaspase-1 activation. Bottom: Loading control tubulin.

FIG. 56 is a western blot of Caspase-1 activation (p10 subunit) inprimary human RPE cells in the presence of iron (III) ammonium citrate.The addition of d4T-ene (Structure 3) (25 μM) blocked iron-inducedCaspase-1 activation. Bottom: Loading control tubulin.

FIG. 57 is a western blot of Caspase-1 activation (p20 subunit) inprimary human RPE cells in the presence of iron (III) ammonium citrate.The addition of 2me-d4T (Structure 4) (25 μM) blocked iron-inducedCaspase-1 activation. Bottom: Loading control tubulin.

FIG. 58 is a western blot of Caspase-1 activation (p20 subunit) inprimary human RPE cells transfected with Alu RNA. The addition ofd4T-ene (Structure 3) and TM-3TC (Structure 8) (25 and 100 μM) blockedAlu-induced Caspase-1 activation. Bottom: Loading control tubulin.

FIG. 59 is a graph of the relative quantity of mtDNA in primary humanRPE cells treated with NRTIs or derivatives (relative to vehicletreatment (DMSO), “No Tx”) at a concentration of 50 μm for all drugs.DNA was collected after four days in cell culture with exposure to drug.Quantitative polymerase chain reaction was performed for mtDNA andnormalized to genomic DNA sequence. Modified versions of d4T that arenot phosphorylated (e.g. me-d4T (FIG. 20), 2 me-d4T (Structure 4)) donot exhibit mtDNA depletion compared to parental NRTI (d4T). TM-3TC isstructure 8. N=3-4/group, error bars are S.E.M.

FIG. 60 is a graph of the relative quantity of mtDNA in primary humanRPE cells treated with NRTIs or derivatives (relative to vehicletreatment (DMSO), “No Tx”) at a concentration of 50 μm for all drugs.DNA was collected after four days in cell culture with exposure to drug.Quantitative polymerase chain reaction was performed for mtDNA andnormalized to genomic DNA sequence. Modified versions of AZT that arenot phosphorylated (me-AZT (Structure 1), N-Me-Me-AZT (Structure 10) donot exhibit mtDNA depletion compared to parental compound (AZT).N=3-4/group, error bars are S.E.M.

FIG. 61 displays a top row of ocular fundus photographs of micereceiving subretinal control empty-vector plasmid (pNull), or AluRNA-expressing plasmid (pAlu), treated with twice daily intraperitonealmodified NRTIs (me-AZT (Structure 1), N-me-me-AZT (Structure 10), or 2me-d4T (Structure 4); 25 mg/kg/administration) or control vehicle, andbottom row RPE flat mounts, stained for intercellular junctions (ZO-1)in red that are disrupted upon Alu RNA expression but that are restoredto healthy RPE morphology/intercellular junctions with modified NRTItreatment. Scale bars 20 μm.

FIG. 62 provides a schematic overview of the synthesis of Formula I(structure C in Scheme 1), Formula IV (structure B), Formula VIII(structure E), Formula X (structure D), and methoxy-d4T (structure A;also FIG. 20).

FIG. 63 is a graph of a lentivirus transduction assay showing thatmodified NRTIs do not block lentivirus replication (in contrast toNRTIs, which blocked lentivirus GFP expression). A GFP-expressinglentivirus was added to 2.5×10³ HeLa cells at multiplicity of infectionof 20 in the presence of NRTIs (AZT, 3TC, d4T), modified NRTIs (me-AZT,structure 1; 2me-AZT, structure 10; TM-3TC, structure 8; me-d4T, FIG.20; 2 me-d4T, structure 4), or control (vehicle). All drugs were addedat a concentration of 10 μM. Cells were imaged for GFP expression 72hours after addition of lentivirus. GFP-positive cells per field of viewwere recorded. n=4-11, error bars S.E.M. Reverse transcription oflentivirus is essential for GFP expression; modified NRTIs should notblock reverse transcriptase, whereas NRTIs are known to block reversetranscriptase.

FIG. 64 is a bar graph showing results of an iGluc cell luminescenceassay, showing inhibition of Caspase-1 activation in a dose-dependentmanner in response to 3TC.

FIG. 65 is a bar graph showing results of an iGluc cell luminescenceassay, showing inhibition of Caspase-1 activation in a dose-dependentmanner in response to 3Me-3TC.

FIG. 66 is a bar graph showing results of an iGluc cell luminescenceassay, showing inhibition of Caspase-1 activation in a dose-dependentmanner in response to 3Et-3TC.

FIG. 67 is a bar graph showing results of an iGluc cell luminescenceassay, showing inhibition of Caspase-1 activation in a dose-dependentmanner in response to AZT.

FIG. 68 is a bar graph showing results of an iGluc cell luminescenceassay, showing inhibition of Caspase-1 activation in a dose-dependentmanner in response to 2Me-AZT.

FIG. 69 is a bar graph showing results of an iGluc cell luminescenceassay, showing inhibition of Caspase-1 activation in a dose-dependentmanner in response to 2Et-AZT.

FIG. 70 is a bar graph showing results of an iGluc cell luminescenceassay, showing inhibition of Caspase-1 activation in a dose-dependentmanner in response to d4T.

FIG. 71 is a bar graph showing results of an iGluc cell luminescenceassay, showing inhibition of Caspase-1 activation in a dose-dependentmanner in response to O-Me N-Et d4T.

FIG. 72 is a bar graph showing results of an iGluc cell luminescenceassay, showing inhibition of Caspase-1 activation in response to Me-d4T.

FIG. 73 is a bar graph showing results of an iGluc cell luminescenceassay, showing inhibition of Caspase-1 activation in a dose-dependentmanner in response to 2Et-d4T.

FIG. 74 includes a series of western blots showing that Alu-RNA-inducedcaspase-1 activation is reduced by 2Et-d4T (25 μM), 2Me-AZT (25 μM),O-Me N-Et d4T (25 μM), d4T-ene (25-100 μM), and TM-3TC (25-100 μM).

FIG. 75 includes a series of western blots showing that iron-inducedcaspase-1 activation is reduced by TM-3TC (25 μM), d4T-ene (25 μM),2Me-d4T (25 μM), Me-AZT (25-100 μM), and 2Me-AZT (25 μM). N=3-4.

FIG. 76 includes a western blot showing that Complement-inducedCaspase-1 activation is reduced by 3TC (25 μM).

FIG. 77 includes a western blot showing that Paraquat-induced Caspase-1activation is reduced by 3TC (25 μM).

FIG. 78 includes a western blot showing that Amyloid beta-inducedCaspase-1 activation is reduced by 3TC (10-25 μM).

FIG. 79 includes data showing choroidal neovascularization in a model of“wet AMD” is reduced by 3TC (0.55 nmol), ABC (0.64 nmol), or AZT (0.55nmol).

FIG. 80 includes data showing choroidal neovascularization (CNV) in amodel of “wet AMD” is reduced by Me-d4T, O-Me-N-Et-d4T, 2Et-AZT or3Et-3TC (0.14 nmol).

FIG. 81 includes fundus photographs of the left eye (top two rows) andthe right eye (bottom two rows) of a first mouse having pre-existing CNVlesions, the first and third row showing time dependent dye leakage fromthe CNV lesions, the second row showing time-dependent dye leakage aftera single intravitreous administration of 125 ng of 3TC, and the fourthrow showing time-dependent dye leakage after a single intravitreousadministration of 62.5 ng of 3-Me-3TC.

FIG. 82 includes fundus photographs of the left eye (top two rows) andthe right eye (bottom two rows) of a second mouse having pre-existingCNV lesions, the first and third row showing time dependent dye leakagefrom the CNV lesions, the second row showing time-dependent dye leakageafter a single intravitreous administration of 125 ng of 3TC, and thefourth row showing time-dependent dye leakage after a singleintravitreous administration of 62.5 ng of 3-Me-3TC.

FIG. 83 includes data showing Alu RNA-induced RPE degeneration in amodel of “dry AMD” is blocked by 3Me-3TC and O-Me N-Et d4T.

FIG. 84 includes data showing pAlu-induced RPE degeneration in a modelof “dry AMD” is blocked by 3Me-3TC.

FIG. 85 includes data showing pAlu-induced RPE degeneration in a modelof “dry AMD” is blocked by 3Et-3TC.

FIG. 86 includes data showing pAlu-induced RPE degeneration in a modelof “dry AMD” is blocked by 2Et-d4T or 2Et-AZT.

FIG. 87 includes data showing poly IC-induced RPE degeneration in amodel of “dry AMD” is blocked by Me-d4T or 2-Me-d4T.

FIG. 88 includes data showing Amyloid beta-induced RPE degeneration in amodel of “dry AMD” by 3TC or K-2 (2Me-d4T).

FIG. 89 includes data showing iron-induced RPE degeneration in a modelof “dry AMD” is blocked by d4T, K-1 (Me-d4T), or K-2 (2Me-d4T).

FIG. 90 includes data showing that d4T, AZT, or 3TC can cause retinaldegeneration.

FIG. 91 includes data showing Kamuvudines are not toxic to the retina.

FIG. 92 includes further data showing Kamuvudines are not toxic to theretina.

FIG. 93 includes data showing that NRTIs (d4T, 3TC, or AZT) reducemitochondrial DNA (mtDNA) while modified NRTIs (Me-d4T, 2Me-d4T, TM-3TCMe-AZT, or 2Me-AZT) do not reduce mtDNA.

FIG. 94 includes data showing that NRTIs (d4T, AZT, or 3TC) inhibit L1retrotransposition but modified NRTIs (Me-d4T, 2Me-d4T, Me-AZT, 2Me-AZT,3Me-3TC, 2Et-d4T, 2Et-AZT, or 3Et-3TC) do not inhibit L1retrotransposition.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The presently-disclosed subject matter is illustrated by specific butnon-limiting examples throughout this description. The examples mayinclude compilations of data that are representative of data gathered atvarious times during the course of development and experimentationrelated to the present invention(s). Each example is provided by way ofexplanation of the present disclosure and is not a limitation thereon.In fact, it will be apparent to those skilled in the art that variousmodifications and variations can be made to the teachings of the presentdisclosure without departing from the scope of the disclosure. Forinstance, features illustrated or described as part of one embodimentcan be used with another embodiment to yield a still further embodiment.

All references to singular characteristics or limitations of the presentdisclosure shall include the corresponding plural characteristic(s) orlimitation(s) and vice versa, unless otherwise specified or clearlyimplied to the contrary by the context in which the reference is made.

All combinations of method or process steps as used herein can beperformed in any order, unless otherwise specified or clearly implied tothe contrary by the context in which the referenced combination is made.

While the terms used herein are believed to be well understood by thoseof ordinary skill in the art, certain definitions are set forth tofacilitate explanation of the presently-disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention(s) belong.

Where reference is made to a URL or other such identifier or address, itunderstood that such identifiers can change and particular informationon the internet can come and go, but equivalent information can be foundby searching the internet. Reference thereto evidences the availabilityand public dissemination of such information.

As used herein, the abbreviations for any protective groups andcompounds, are, unless indicated otherwise, in accord with their commonusage, recognized abbreviations, or the IUPAC-IUB Commission onBiochemical Nomenclature (see, Biochem. (1972) 11(9):1726-1732).

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a cell” includes aplurality of such cells, and so forth.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as reaction conditions, and so forth usedin the specification and claims are to be understood as being modifiedin all instances by the term “about”. Accordingly, unless indicated tothe contrary, the numerical parameters set forth in this specificationand claims are approximations that can vary depending upon the desiredproperties sought to be obtained by the presently-disclosed subjectmatter.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, insome embodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

As used herein, ranges can be expressed as from “about” one particularvalue, and/or to “about” another particular value. It is also understoodthat there are a number of values disclosed herein, and that each valueis also herein disclosed as “about” that particular value in addition tothe value itself. For example, if the value “10” is disclosed, then“about 10” is also disclosed. It is also understood that each unitbetween two particular units are also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

As used herein, “optional” or “optionally” means that the subsequentlydescribed event or circumstance does or does not occur and that thedescription includes instances where said event or circumstance occursand instances where it does not. For example, an optionally variantportion means that the portion is variant or non-variant.

The term “physiologically functional derivative” means anypharmaceutically acceptable derivative of a compound of the presentdisclosure. For example, an amide or ester of a compound of formula (I)or of a compound of formula (II), which upon administration to asubject, particularly a mammal, is capable of providing, either directlyor indirectly, a compound of the present disclosure of an activemetabolite thereof.

The terms “treatment” or “treating” refer to the medical management of asubject with the intent to cure, ameliorate, stabilize, or prevent acondition or disorder (e.g., retinal degradation). This term includesactive treatment, that is, treatment directed specifically toward theimprovement of a condition, and also includes causal treatment, that is,treatment directed toward removal of the cause of the associatedcondition. In addition, this term includes palliative treatment, thatis, treatment designed for the relief of symptoms rather than the curingof the condition; preventative treatment, that is, treatment directed tominimizing or partially or completely inhibiting the development ofsymptoms or disorders of the associated condition; and supportivetreatment, that is, treatment employed to supplement another specifictherapy directed toward the improvement of the associated disease,pathological condition, or disorder.

With regard to administering the compound, the term “administering”refers to any method of providing a composition and/or pharmaceuticalcomposition thereof to a subject. Such methods are well known to thoseskilled in the art and include, but are not limited to, oraladministration, transdermal administration, administration byinhalation, nasal administration, topical administration, intravaginaladministration, ophthalmic administration, intraaural administration,intracerebral administration, rectal administration, and parenteraladministration, including injectable such as intravenous administration,intra-arterial administration, intramuscular administration,subcutaneous administration, intravitreous administration, including viaintravitreous sustained drug delivery device, intracameral (intoanterior chamber) administration, suprachoroidal injection, subretinaladministration, Subconjunctival injection, sub-Tenon's administration,peribulbar administration, Transscleral drug delivery, administrationvia topical eye drops, and the like. Administration can be continuous orintermittent. In various aspects, a preparation can be administeredtherapeutically; that is, administered to treat an existing disease orcondition (e.g., exposure to OP compounds). In further various aspects,a preparation can be administered prophylactically; that is,administered for prevention of a disease or condition.

The term “effective amount” refers to an amount that is sufficient toachieve the desired result or to have an effect on an undesiredcondition. For example, a “therapeutically effective amount” refers toan amount that is sufficient to achieve the desired therapeutic resultor to have an effect on undesired symptoms, but is generallyinsufficient to cause adverse side effects. The specific therapeuticallyeffective dose level for any particular patient will depend upon avariety of factors including the disorder being treated and the severityof the disorder; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration; the route of administration; the rate of excretion ofthe specific compound employed; the duration of the treatment; drugsused in combination or coincidental with the specific compound employedand like factors well known in the medical arts. For example, it is wellwithin the skill of the art to start doses of a compound at levels lowerthan those required to achieve the desired therapeutic effect and togradually increase the dosage until the desired effect is achieved. Ifdesired, the effective daily dose can be divided into multiple doses forpurposes of administration. Consequently, single dose compositions cancontain such amounts or submultiples thereof to make up the daily dose.The dosage can be adjusted by the individual physician in the event ofany contraindications. Dosage can vary, and can be administered in oneor more dose administrations daily, for one or several days. Guidancecan be found in the literature for appropriate dosages for given classesof pharmaceutical products. In further various aspects, a preparationcan be administered in a “prophylactically effective amount”; that is,an amount effective for prevention of a disease or condition.

The terms “subject” or “subject in need thereof” refer to a target ofadministration, which optionally displays symptoms related to aparticular disease, condition, disorder, or the like. The subject(s) ofthe herein disclosed methods can be human or non-human (e.g., primate,horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, rodent, andnon-mammals). The term “subject” does not denote a particular age orsex. Thus, adult and newborn subjects, as well as fetuses, whether maleor female, are intended to be covered. The term “subject” includes humanand veterinary subjects.

As will be recognized by one of ordinary skill in the art, the terms“suppression,” “suppressing,” “suppressor,” “inhibition,” “inhibiting”or “inhibitor” do not refer to a complete elimination of angiogenesis inall cases. Rather, the skilled artisan will understand that the term“suppressing” or “inhibiting” refers to a reduction or decrease inangiogenesis. Such reduction or decrease can be determined relative to acontrol. In some embodiments, the reduction or decrease relative to acontrol can be about a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% decrease.

In some exemplary embodiments, the presently-disclosed subject matterincludes methods for treating retinal damage and/or retinaldegeneration. Indeed, some methods of the present disclosure compriseadministering to a subject in need thereof an effective amount of acomposition for treating retinal damage and/or degradation.

In some embodiments the composition comprises a nucleoside and/or anucleoside reverse transcriptase inhibitor (NRTI). Further, in someembodiments, the composition is a pharmaceutical composition comprisinga nucleoside and/or a NRTI compound as well as a pharmaceuticallyacceptable carrier.

1001211 As discussed herein, in some exemplary methods of the presentdisclosure, the administered composition is a composition comprising anucleoside and/or NRTI. Thus, exemplary compositions are comprised ofcompounds including, but not limited to, stavudine (d4T), lamivudine(3TC), cordycepin, azidothymidine (AZT), abacavir (ABC), chemicalderivatives thereof (e.g., methoxy-derivatives to abrogatephosphorylation), and the like. Other possible compounds include, forexample, those described in U.S. Pat. No. 6,514,979 to Heredia et al.Those of ordinary skill in the art will also recognize furthernucleosides and/or NRTIs, as described herein, that can be used in thecompositions and methods of this disclosure.

In some embodiments a method of the present disclosure comprisesinhibiting activation of one or more physiological processes by Alu RNA.As disclosed herein, Alu RNA (including Alu repeat RNA in human cellsand B1 and B2, Alu-like element repeat RNAs) increases are associatedwith cells that are associated with certain conditions of interest. Forexample, an Alu RNA increase is associated with the retinal pigmentepithelium (RPE) cells of eyes with geographic atrophy. This increase ofAlu RNA induces the death of RPE cells. Methods and compositionsdisclosed herein can treat RPE degradation, thereby treating conditionsassociated with such cell death.

In some embodiments, a method of the present disclosure comprisesinhibiting the activation of at least one inflammasome. In certainembodiments, the at least one inflammasome is selected from an NLRP3inflammasome, a 1L-1beta inflammasome, and a combination thereof. Insome embodiments, the inhibiting one or more inflammasomes of a cellincludes administering an inhibitor (composition) to the cell and/or toa subject, wherein the cell is the cell of a subject. For compositionscomprising an inhibitor, an inhibitor as described herein can be, forexample, a polypeptide inhibitor (including an oligonucleotideinhibitor), a small molecule inhibitor, and/or an siRNA inhibitor.

Moreover, some exemplary methods of administering the presentcomposition(s) can inhibit inflammation by LPS/ATP, inflammasomeactivation by LPS/ATP, inflammasome activation by Alu RNA, and/ornigericin-induced inflammasome activation. Exemplary methods can alsotreat retinal degradation and/or other retinal damage by reducingmitochondrial reactive oxygen species, particularly as caused by Alu RNAexpression, by blocking entry via the P2X7 receptor, and/or by reducingATP-induced cell permeability.

In some embodiments, a method of the present disclosure comprisestreating retinal damage by inhibiting a particular action in a cell. Insome embodiments, the cell is selected from an RPE cell, a retinalphotoreceptor cell, or a choroidal cell. In some embodiments, the cellis an RPE cell. In some embodiments, the cell is the cell of a subject.In some embodiments, the cell is a cell of a subject having, suspectedof having, or at risk of having a condition of interest. In someembodiments, the cell is a cell of a subject having, suspected ofhaving, or at risk of having age-related macular degeneration. In someembodiments, the cell is a cell of a subject having, suspected ofhaving, or at risk of having geographic atrophy. In some embodiments,the cell is a cell of a subject having, suspected of having, or at riskof having geographic atrophy and the cell is an RPE cell. In someembodiments, a subject having age-related macular degeneration can betreated using methods and compositions as disclosed herein.

Thus, as used herein with reference to a polypeptide being inhibited,“of a cell” refers to a polypeptide that is inside the cell (inside thecell membrane), on the cell (in the cell membrane, presented on the cellmembrane, otherwise on the cell), or outside of a cell, but insofar asthe polypeptide is outside of the cell, it is in the extracellularmilieu such that one of ordinary skill in the art would recognize thepolypeptide as being associated with the cell. For example, VDAC1,VDAC2, caspase-8, NFκB, or a polypeptide of an inflammasome (e.g.,NLRP3, PYCARD, caspase-1) could be in the cell. For another example,NLRP3 could be in the cell or on the cell.

As described herein, the presently-disclosed subject matter furtherincludes pharmaceutical compositions comprising the compounds describedherein together with a pharmaceutically acceptable carrier.

The term “pharmaceutically acceptable carrier” refers to sterile aqueousor nonaqueous solutions, dispersions, suspensions or emulsions, as wellas sterile powders for reconstitution into sterile injectable solutionsor dispersions just prior to use. Proper fluidity can be maintained, forexample, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions andby the use of surfactants. These compositions can also contain adjuvantssuch as preservatives, wetting agents, emulsifying agents and dispersingagents. Prevention of the action of microorganisms can be ensured by theinclusion of various antibacterial and antifungal agents such asparaben, chlorobutanol, phenol, sorbic acid and the like. It can also bedesirable to include isotonic agents such as sugars, sodium chloride andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the inclusion of agents, such as aluminummonostearate and gelatin, which delay absorption. Injectable depot formsare made by forming microencapsule matrices of the drug in biodegradablepolymers such as polylactide-polyglycolide, poly(orthoesters) andpoly(anhydrides). Depending upon the ratio of drug to polymer and thenature of the particular polymer employed, the rate of drug release canbe controlled. Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues. The injectable formulations can be sterilized, forexample, by filtration through a bacterial-retaining filter or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable media just prior to use. Suitable inertcarriers can include sugars such as lactose.

Suitable formulations include aqueous and non-aqueous sterile injectionsolutions that can contain antioxidants, buffers, bacteriostats,bactericidal antibiotics and solutes that render the formulationisotonic with the bodily fluids of the intended recipient; and aqueousand non-aqueous sterile suspensions, which can include suspending agentsand thickening agents.

The compositions can take such forms as suspensions, solutions oremulsions in oily or aqueous vehicles, and can contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The formulations can be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and can be stored in a frozen orfreeze-dried (lyophilized) condition requiring only the addition ofsterile liquid carrier immediately prior to use.

For oral administration, the compositions can take the form of, forexample, tablets or capsules prepared by a conventional technique withpharmaceutically acceptable excipients such as binding agents (e.g.,pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talcor silica); disintegrants (e.g., potato starch or sodium starchglycollate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets can be coated by methods known in the art.

Liquid preparations for oral administration can take the form of, forexample, solutions, syrups or suspensions, or they can be presented as adry product for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional techniqueswith pharmaceutically acceptable additives such as suspending agents(e.g., sorbitol syrup, cellulose derivatives or hydrogenated ediblefats); emulsifying agents (e.g. lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations can alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration can be suitablyformulated to give controlled release of the active compound. For buccaladministration the compositions can take the form of tablets or lozengesformulated in conventional manner.

The compositions can be formulated as eye drops. For example, thepharmaceutically acceptable carrier may comprise saline solution orother substances used to formulate eye drop, optionally with otheragents. Thus, eye drop formulations permit for topical administrationdirectly to the eye of a subject.

The compositions can also be formulated as a preparation forimplantation or injection. Thus, for example, the compounds can beformulated with suitable polymeric or hydrophobic materials (e.g., as anemulsion in an acceptable oil) or ion exchange resins, or as sparinglysoluble derivatives (e.g., as a sparingly soluble salt). The compoundscan also be formulated in rectal compositions, creams or lotions, ortransdermal patches.

The presently-disclosed subject matter further includes a kit that caninclude a compound or pharmaceutical composition as described herein,packaged together with a device useful for administration of thecompound or composition. As will be recognized by those or ordinaryskill in the art, the appropriate administration-aiding device willdepend on the formulation of the compound or composition that isselected and/or the desired administration site. For example, if theformulation of the compound or composition is appropriate for injectionin a subject, the device could be a syringe. For another example, if thedesired administration site is cell culture media, the device could be asterile pipette.

Moreover, NRTIs of the present disclosure are a diverse, widely used,inexpensive class of small molecules, with extensive pharmacokinetic andsafety data collected over the past several decades of human use; NRTIsare therefore ripe for drug repurposing. As such, the present disclosureprovides a novel and broadly applicable basis for use of one or moreNRTIs by addressing major unmet medical needs.

As briefly described above, age-related macular degeneration is adisease that affects tens of millions of people worldwide, and there isno effective treatment for AMD (Ambati and Fowler, 2012). Similarly,graft-versus host disease is the major obstacle preventing successfultissue transplant (Ferrara et al., 2009); and sterile liver inflammationis a major contributor to drug-induced liver injury and steatohepatitis,a major determinant of fibrosis and carcinogenesis (Kubes and Mehal,2012). Thus, some methods and/or compounds of the present disclosure areintended to treat age-related macular degeneration, graft-versus hostdisease, and/or sterile liver inflammation by administering, in someembodiments, a compound comprising at least one NRTI, as provided in thepresent disclosure.

Since inflammasome inhibition by NRTIs can be achieved withoutphosphorylation of a particular NRTI, the use of me-d4T or otherphosphorylation-incompetent nucleoside analogs, as provided herein,should avoid therapeutic-limiting toxicities associated withNRTI-triphosphate-mediated polymerase inhibition (Lewis et al., 2003).Accordingly, in some embodiments, the present disclosure is directed tomethods for treating retinal disease by administering me-d4T or anotherphosphorylation-incompetent nucleoside analog to a subject in needthereof.

Further, in certain embodiments, the present disclosure provides methodsfor treating retinal damage, comprising: administering an effectiveamount of a compound or composition to a subject in need thereof,wherein the composition comprises a compound as disclosed herein orcombinations thereof.

In some embodiments, the presently disclosed subject matter providesmethods for protecting an RPE cell, a retinal photoreceptor cell, achoroidal cell, or a combination thereof, comprising at least the stepof administering an effective amount of a compound or composition to asubject in need thereof, wherein the composition comprises a compound asdisclosed herein or combinations thereof.

In some embodiments, the presently disclosed subject matter providesmethods for conditions associated with retinal damage and/ordegradation, which involve administering an effective amount of acompound or composition to a subject in need thereof, wherein thecomposition comprises a compound as disclosed herein or combinationsthereof.

In some embodiments, the presently disclosed subject matter providesmethods for treating age related macular degeneration (AMD), whichinvolve administering an effective amount of a compound or compositionto a subject in need thereof, wherein the composition comprises acompound as disclosed herein or combinations thereof. In someembodiments the AMD is wet AMD. In some embodiments the AMD is dry AMD.

As described herein, the present inventors have found that nucleosidereverse transcriptase inhibitors, which are FDA-approved for thetreatment of HIV and HBV, were surprisingly found to be effective inmouse models of dry and wet age-related macular degeneration (AMD)(Fowler et al. Science 2014). However, some of the NRTIs tested by thepresent inventors (i.e. d4T, AZT) cause undesirable side effects inpatients, which is thought to occur due to off-target effects on DNApolymerase-gamma, which leads to mitochondrial depletion. If NRTIs areto be used to treat chronic diseases such as AMD, long-term polymeraseinhibition with NRTIs could hinder their clinical translation.

The present inventors designed a novel methoxy-modified version of d4T(Fowler et al. Science 2014). However, the synthesis of me-d4T waslaborious (over 10 steps). Furthermore, other NRTIs such as AZT and 3TCalso blocked mouse models of dry and wet AMD (Fowler et al. Science2014; Mizutani et al. IOVS 2015, in press), although it is not knownwhether modified versions of these drugs are also effective in thesemodels. Accordingly, the present-disclosed subject matter includesunique compounds useful for the indications as disclosed herein, withoutthe drawbacks of previously-known compounds.

The presently-disclosed subject matter provides, in certain embodiments,a compound having the structure

or a pharmaceutically acceptable salt thereof, wherein

R₁ is selected from covalent bond, H, alkyl, substituted alkyl, branchedalkyl, alkylene, acyl, alkoxyl, acyloxyl, and acylamino; in someembodiments, R₁ is selected from ethyl, butyl, propyl, 2-methylpropyl,and t-butyl; and in some embodiments R₁ is selected from covalent bond,H and —CH₃;

R₂ is selected from H, alkyl, substituted alkyl, branched alkyl,alkylene, acyl, alkoxyl, acyloxyl, and acylamino; in some embodiments,R₂ is selected from ethyl, butyl, propyl, 2-methylpropyl, and t-butyl;and in some embodiments, R₂ is —N(R₃)₂, where each R₃ is independentlyselected from alkyl, substituted alkyl, branched alkyl, alkylene, acyl,alkoxyl, acyloxyl, and acylamino, and in sonic embodiments, each R₃ isindependently selected from H, CH₃, ethyl, butyl, t-butyl, isobutyl,propyl, 2-methylpropyl, isopropyl, pentyl, and hexyl;

R₄ is selected from H, alkyl, substituted alkyl, branched alkyl,alkylene, acyl, alkoxyl, acyloxyl, and acylamino; and in someembodiments, R₄ is selected from —CH₃ and ethyl;

R₅ is selected from C, CH, and S;

R₆ is selected from H, alkyl, substituted alkyl, branched alkyl,alkylene, acyl, alkoxyl, acyloxyl, and acylamino; in some embodiments,R₆ is selected from ethyl, butyl, propyl, 2-methylpropyl, and t-butyl;and in some embodiments, R₆ is selected from —N═N⁺═NH and N₃; and

R₇ is H, alkyl, substituted alkyl, branched alkyl, alkylene, acyl,alkoxyl, acyloxyl, and acylamino; in some embodiments, R₇ is selectedfrom ethyl, butyl, propyl, 2-methylpropyl, and t-butyl; in someembodiments, R₇ is selected from —CH₂—O—CH₃, —CH₃, ═CH₂, —CH₂—NH₂, and—CH₂—N═N⁺═NH; and in some embodiments, R₇ is —CH₂—O—R₈, wherein R₈ isselected from alkyl, substituted alkyl, alkylene, acyl, alkoxyl,acyloxyl, and acylamino, and in some embodiments, R₈ is selected from—CH₃, ethyl, propyl, 2-methylpropyl, isopropyl, butyl, t-butyl,isobutyl, pentyl, hexyl.

As used herein the term “alkyl” refers to C1-20 inclusive, linear (i.e.,“straight-chain”), branched, or cyclic, saturated or at least partiallyand in some cases fully unsaturated (i.e., alkenyl and alkynyl)hydrocarbon chains, including for example, methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tort-butyl, pentyl, hexyl, octyl, ethenyl,propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl,methylpropynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenylgroups. “Branched” refers to an alkyl group in which a lower alkylgroup, such as methyl, ethyl or propyl, is attached to a linear alkylchain. “Lower alkyl” refers to an alkyl group having 1 to about 8 carbonatoms (i.e., a C1-8 alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbonatoms. “Higher alkyl” refers to an alkyl group having about 10 to about20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20carbon atoms. In certain embodiments, “alkyl” refers, in particular, toC1-8 straight-chain alkyls. In other embodiments, “alkyl” refers, inparticular, to C1-8 branched-chain alkyls.

Alkyl groups can optionally be substituted (a “substituted alkyl”) withone or more alkyl group substituents, which can be the same ordifferent. The term “alkyl group substituent” includes but is notlimited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl,aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio,carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There can be optionallyinserted along the alkyl chain one or more oxygen, sulfur or substitutedor unsubstituted nitrogen atoms, wherein the nitrogen substituent ishydrogen, lower alkyl (also referred to herein as “alkylaminoalkyl”), oraryl.

Thus, as used herein, the term “substituted alkyl” includes alkylgroups, as defined herein, in which one or more atoms or functionalgroups of the alkyl group are replaced with another atom or functionalgroup, including for example, alkyl, substituted alkyl, halogen, aryl,substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino,dialkylamino, sulfate, and mercapto.

Further, as used herein, the terms alkyl and/or “substituted alkyl”include an “allyl” or an “allylic group.” The terms “allylic group” or“allyl” refer to the group CH2HC═CH2 and derivatives thereof formed bysubstitution. Thus, the terms alkyl and/or substituted alkyl includeallyl groups, such as but not limited to, allyl, methylallyl,di-methylallyl, and the like. The term “allylic position” or “allylicsite” refers to the saturated carbon atom of an allylic group. Thus, agroup, such as a hydroxyl group or other substituent group, attached atan allylic site can be referred to as “allylic.”

“Alkylene” refers to a straight or branched bivalent aliphatichydrocarbon group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3,4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbonatoms. The alkylene group can be straight, branched or cyclic. Thealkylene group also can be optionally unsaturated and/or substitutedwith one or more “alkyl group substituents.” There can be optionallyinserted along the alkylene group one or more oxygen, sulfur orsubstituted or unsubstituted nitrogen atoms (also referred to herein as“alkylaminoalkyl”), wherein the nitrogen substituent is alkyl aspreviously described. Exemplary alkylene groups include methylene(—CH₂—); ethylene (—CH₂—CH₂—); propylene (—(CH2)3-); cyclohexylene(—C6H10-); —CH═CH—CH═CH—; —CH═CH—CH2-; —(CH2)q-N(R)—(CH2)r-; whereineach of q and r is independently an integer from 0 to about 20, e.g., 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20, and R is hydrogen or lower alkyl; methylenedioxyl (—O—CH2-O—); andethylenedioxyl (—O—(CH2)2-O—). An alkylene group can have about 2 toabout 3 carbon atoms and can further have 6-20 carbons.

As used herein, the term “acyl” refers to an organic acid group whereinthe OH of the carboxyl group has been replaced with another substituent(i.e., as represented by RCO—, wherein R is an alkyl or an aryl group asdefined herein). As such, the term “acyl” specifically includes arylacylgroups, such as an acetylfuran and a phenacyl group. Specific examplesof acyl groups include acetyl and benzoyl.

“Alkoxyl” or “alkoxyalkyl” refer to an alkyl-O— group wherein alkyl isas previously described. The term “alkoxyl” as used herein can refer toC1-20 inclusive, linear, branched, or cyclic, saturated or unsaturatedoxo-hydrocarbon chains, including, for example, methoxyl, ethoxyl,propoxyl, isopropoxyl, butoxyl, t butoxyl, and pentoxyl.

“Acyloxyl” refers to an acyl-O— group wherein acyl is as previouslydescribed.

“Acylamino” refers to an acyl-NH— group wherein acyl is as previouslydescribed.

The term “amino” refers to the —NH2 group.

A dashed line representing a bond in a cyclic ring structure indicatesthat the bond can be either present or absent in the ring. That is adashed line representing a bond in a cyclic ring structure indicatesthat the ring structure is selected from the group consisting of asaturated ring structure, a partially saturated ring structure, and anunsaturated ring structure.

Moreover, in some embodiments, the present disclosure is directed to thecompounds of, pharmaceutical compositions including compounds of,synthesis of, and/or use of one or more of the compounds disclosedhereinbelow.

In some embodiments, the presently disclosed compound has the structureof any one of Formula I-XIV or a pharmaceutically acceptable saltthereof.

(I) Me-AZT:1-[(5R)-2-(methoxymethyl)-5-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)oxolan-3-yl]triaza-1,2-dien-2-ium(also referred to herein as Kamuvudine 5)

(II) deoxy-methyl-d4T:5-methyl-1-[2R,5R)-5-methyl-2,5-dihydrofuran-2-yl]-1,2,3,4-tetrahydropyrimidine-2,4-dione

(III) methylene d4T:5-methyl-1-[2R)-5-methylidene-2,5-dihydrofuran-2-yl]-1,2,3,4-tetrahydropyrimidine-2,4-dione

(IV) 2Me-d4T:1-[(2R)-5-(methoxymethyl)-2,5-dihydrofuran-2-yl]-3,5-dimethyl-1,2,3,4-tetrahydropyrimidine-2,4-dione(also referred to herein “Kamuvudine 2”)

(V) deoxy-amino-d4T:1-[(2R)-5-(aminomethyl)-2,5-dihydrofuran-2-yl]-5-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione

(VI) Me-3TC:4-amino-1-[(2R,5S)-2-(methoxymethyl)-1,3-oxathiolan-5-yl]-1,2-dihydropyrimidin-2-one

(VII) deamino-Me-3TC:1-[(2R,5S)-2-(methoxymethyl)-1,3-oxathiolan-5-yl]-1,2,3,4-tetrahydropyrimidine-2,4-dione

(VIII) 3Me-3TC:4-(dimethylamino)-1-[(2R,5S)-2-(methoxymethyl)-1,3-oxathiolan-5-yl]-1,2-dihydropyrimidin-2-one(also referred to herein as Kamuvudine 6 and TM-3TC)

(IX) Azido-d4T:1-{[(2S,5R)-5-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)-2,5-dihydrofuran-2-yl]methyl}triaza-1,2-dien-2-ium

(X) 2Me-AZT:1-[(2R,4S,5S)-4-azido-5-(methoxymethyl)oxolan-2-yl]-3,5-dimethyl-1,2,3,4-tetrahydropyrimidine-2,4-dione(also referred to herein as Kamuvudine 4)

(XI) O-Me N-Et d4T:3-ethyl-1-[(2R,5S)-5-(methoxymethyl)-2,5-dihydrofuran-2-yl]-5-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione(also referred to herein as Kamuvudine 3)

(XII) 2Et-AZT: 1-[(2R,4S,5S)-4-azido-5-(ethoxymethyl)oxolan-2-yl]-3-ethyl-5-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione(also referred to herein as Kamuvudine 8)

(XIII) 2Et-d4T: 1-[(2R,5S)-5-(ethoxymethyl)-2,5-dihydrofuran-2-yl]-3-ethyl-5-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione(also referred to herein as Kamuvudine 7)

(XIV) 3Et-3TC:4-(diethylamino)-1-[2-(ethoxymethyl)-1,3-oxathiolan-5-yl]-1,2-dihydropyrimidin-2-one(also referred to herein as Kamuvudine 9)

In some embodiments, the presently disclosed compound has the structureof any one of the following compounds, or a pharmaceutically acceptablesalt thereof:

In some embodiments, the presently disclosed compound has the structureof any one of the following compounds, or a pharmaceutically acceptablesalt thereof:

In some embodiments, the presently disclosed compound has the structureof any one of the following compounds, or a pharmaceutically acceptablesalt thereof:

Further, the present disclosure provides uses of the compounds disclosedherein, or any combination thereof, in the preparation or manufacture ofa pharmaceutical composition, such as a drug and/or medicine, especiallya composition for the treatment of retinal damage and/or retinaldegeneration in a mammal. In some embodiments, the present disclosureprovides a pharmaceutical composition comprising the compounds asdisclosed herein, any salt, particularly any pharmaceutically acceptablesalt, any solvate, and/or any physiological derivative thereof, togetherwith a pharmaceutically acceptable carrier.

In certain embodiments, the methods and compositions of the presentdisclosure inhibit graft-versus-host disease, chronic pain,proliferative vitreoretinopathy, glaucoma, rheumatoid arthritis,multiple sclerosis, bipolar disorder, major depressive disorder, renalfibrosis, nephritis, pulmonary fibrosis, Huntington's disease,osteoporosis, chronic lymphocytic leukemia, anxiety disorders, pulmonarytuberculosis, osteoporosis in post-menopausal women and fracturepatients, systemic lupus erythematosus, discoid lupus erythematosus,chronic inflammatory and neuropathic pain, autosomal dominant polycystickidney disease, spinal cord injury, Alzheimer's disease, neuropathicpain, hypertension, varicose veins, type I diabetes, type II diabetes,gout, autoimmune hepatitis, graft vascular injury, atherosclerosis,thrombosis, metabolic syndrome, salivary gland inflammation, traumaticbrain injury, ischemic heart disease, ischemic stroke, Parkinson'sdisease, melanoma, neuroblastoma, prostate, breast, skin, and thyroidcancers, tubular early gastric cancer, neuroendocrine cancer, mucoidcolon cancer, colon cancer; high-grade urothelial carcinoma, kidneyclear cell carcinoma, undifferentiated ovary carcinoma, papillaryintracystic breast carcinoma, gram negative sepsis, infectiousPseudomonas aeruginosa, Vibrio cholera, Legionella spp., Francisellaspp., and Leishmania spp. Chlamydia spp., cryopyrinopathies; keratitis,acne vulgaris, Crohn's disease, ulcerative colitis, irritable bowelsyndrome, insulin resistance, obesity, hemolytic-uremic syndrome,polyoma virus infection, immune complex renal disease, acute tubularinjury, lupus nephritis, familial cold autoinflammatory syndrome,Muckle-Wells syndrome and neonatal onset multisystem inflammatorydisease, chronic infantile neurologic cutaneous and articularautoinflammatory diseases, renal ischemia-perfusion injury,glomerulonephritis, cryoglobulinemia, systemic vasculitides, IgAnephropathy, malaria, helminth parasites, septic shock, allergic asthma,hay fever, chronic obstructive pulmonary disease, drug-induced lunginflammation, contact dermatitis, leprosy, Burkholderia cenocepaciainfection, respiratory syncitial virus infection, psoriasis,scleroderma, reactive arthritis, cystic fibrosis, syphilis, Sjögren'ssyndrome, inflammatory joint disease, non-alcoholic fatty liver disease,cardiac surgery (peri-/post-operative inflammation), acute and chronicorgan transplant rejection, acute and chronic bone marrow transplantrejection, tumor angiogenesis, amyotrophic lateral sclerosis, autismspectrum disorder (e.g., through Kamuvudine blockade of P2X7, as shownin mouse models of autism), and/or any combination thereof.

Moreover, in some embodiments, the present disclosure provides thatnon-canonical NRTI function, independent of chain termination, preventsP2X7-dependent blindness, graft-versus-host disease and/or sterileinflammation. Accordingly, the present disclosure is directed, incertain embodiments, to methods of preventing P2X7-dependent blindness,graft-versus-host disease and/or inflammation in a subject byadministering an effective amount of at least one NRTI, as describedherein, to subject in need thereof.

Further, in certain embodiments, the methods and compositions of thepresent disclosure inhibit (i) inflammasome activation by Alu RNAassociated with a cell; (ii) inflammation by LPS/ATP, (iii) inflammasomeactivation by LPS/ATP, (iv) nigericin-induced inflammasome activation,and/or combinations thereof. And in some embodiments, the inflammasomeis selected from the group consisting of a NLRP3 inflammasome and/or a1L-1beta inflammasome. Additionally, some embodiments of the methods ofthe present disclosure may include, for example, the steps of (i)blocking entry via a P2X7 receptor associated with a cell; (ii) reducingmitochondrial reactive oxygen species caused by Alu RNA expression;and/or (iii) reducing ATP-induced cell permeability of a cell. And acell contemplated in the present disclosure may include, for example, anRPE cell, a retinal photoreceptor cell, a choroidal cell, or anycombination thereof.

Further, NRTIs are mainstay therapeutics for HIV, and they blockretrovirus replication. Alu RNA, an endogenous retroelement that alsorequires reverse transcriptase (RT) for its life cycle, activates theNLRP3 inflammasome to cause cell death of the retinal pigment epitheliumin geographic atrophy, which is the untreatable form of age-relatedmacular degeneration that blinds millions of individuals. Moreover, theinventors of the present disclosure have found that NRTIs, as a class,are novel inhibitors of the NLRP3 inflammasome. And, surprisingly, thiseffect is independent of reverse transcriptase inhibition.

The details of one or more embodiments of the presently-disclosedsubject matter are set forth in this document. Modifications toembodiments described in this document, and other embodiments, will beevident to those of ordinary skill in the art after a study of theinformation provided in this document. The information provided in thisdocument, and particularly the specific details of the describedexemplary embodiments, is provided primarily for clearness ofunderstanding and no unnecessary limitations are to be understoodtherefrom. In case of conflict, the specification of this document,including definitions, will control.

The presently-disclosed subject matter is further illustrated by thefollowing specific but non-limiting examples. The following examples mayinclude compilations of data that are representative of data gathered atvarious times during the course of development and experimentationrelated to the present invention.

EXAMPLES Example 1

The inventors of the present disclosure have found that the NRTIs d4T,AZT, ABC, and 3TC block Caspase 1 activation by Alu RNA, as does5′-methoxy-d4T, which does not inhibit reverse transcriptase. Further,the present inventors have found that AZT is not phosphorylated inthymidine kinase-deficient cells but still blocks LPS/ATP-inducedinterleukin-1 beta secretion; that NRTIs block P2X7-dependent YOPRO-1dye uptake and mouse models of geographic atrophy, graft-versus-hostdisease, and sterile liver inflammation; and that NRTIs are novelinhibitors of the NLRP3 inflammasome independent of canonical reversetranscriptase inhibition. Accordingly, NRTIs are ripe for drugrepurposing in a variety of P2X7-driven diseases.

NRTIs were first discovered to be anti-viral compounds in 1974 (Ostertaget al., 1974), and are widely used to treat human immunodeficiency virus(HIV). The canonical mechanism of action of NRTIs is via chaintermination of DNA synthesis from a viral RNA template, therebyinterfering with the viral life cycle of reverse transcriptase-dependentviruses.

Age-related macular degeneration (AMD) is a leading cause of blindnessin the elderly worldwide (Ambati et al., 2003; Ambati and Fowler, 2012).In the more prevalent and untreatable dry form of AMD, overabundance ofnon-coding Alu RNAs causes blindness by inducing cell death of theretinal pigment epithelium (Dridi et al., 2012; Kaneko et al., 2011;Tarallo et al., 2012). Alu sequences are non-coding retrotransposonsthat, like HIV, rely on reverse transcriptase for their life cycle(Batzer and Deininger, 2002; Dewannieux et al., 2003).

Alu RNA mediates RPE cell death via activation of Caspase 1 and theNLRP3 inflammasome (Tarallo et al., 2012). The present disclosureprovides that a reverse transcriptase inhibitor, such as stavudine (d4T;2′3′ dideoxythymidine; Zerit, Bristol-Myers Squibb), which isFDA-approved for the treatment of HIV, prevents Caspase 1 cleavage toits active 20 kDa form (Hentze et al., 2003; Yamin et al., 1996) inprimary human (FIG. 34) and mouse RPE cells (FIG. 44) without reducingAlu RNA levels (FIG. 45). Further, the present disclosure shows that d4Talso blocks phosphorylation of IRAK4, a kinase downstream of the MyD88adaptor that mediates Alu-induced RPE cell death (Tarallo et al., 2012),in human and mouse RPE cells (FIG. 34 and FIG. 44). The inventors of thepresent disclosure have also found that other NRTIs, including theanti-HIV drugs azidothymidine (AZT; 3′-azido-2′,3′-dideoxythymidine;Retrovir, ViiV Healthcare), lamivudine (3TC; 2′3′ dideoxycytidine;Zeffix, GlaxoSmithKline) and abacavir (ABC; a di-deoxyguanosine analog;Ziagen, ViiV Healthcare), also block Caspase-1 cleavage induced by AluRNA (FIG. 35).

Additionally, the present disclosure provides that d4T and AZT preventRPE degeneration in the Alu RNA-induced mouse model of dry AMD. (Kanekoet al., 2011; Tarallo et al., 2012) Moreover, it has been found thatmice receiving daily oral administration of d4T blocked RPE degenerationafter sub-retinal injection of a plasmid expressing Alu RNA (FIG. 36),as did intraperitoneal administration of AZT (FIG. 37).

In order to test whether reverse transcriptase inhibition was requiredfor inflammasome blockade by d4T, a 5′ O-methyl-modified version of d4T(5′-OCH3-d4T; me-d4T) was synthesized (FIG. 20; FIG. 25, FIG. 36, FIG.27, FIG. 28). Accordingly, in some embodiments, the present disclosureis directed to methods for synthesizing a 5′ O-methyl-modified versionof d4T as provided herein.

Only the triphosphate version of nucleoside analogs inhibits reversetranscriptase; the methyl modification at the 5′ position preventsphosphorylation and thus formation of nucleoside triphosphate (Nykanenet al., 2001). Accordingly, like d4T, me-d4T also blocks Caspase-1activation in human RPE cells (FIG. 21).

The present inventors have confirmed that me-d4T does not inhibitreverse transcriptase: and, in contrast to unmodified d4T, me-d4T doesnot block lentivirus replication (FIG. 22). Also, the triphosphatemetabolite of di-deoxy nucleoside analogs caused depletion ofmitochondrial DNA; and consistent with the idea that me-d4T is notphosphorylated, it has been found that d4T, but not me-d4T reduces mtDNAlevels. (FIG. 23). Me-d4T also prevents Alu-induced RPE degeneration inmice (FIG. 24). These data indicate that d4T can block Caspase-1activation and RPE degeneration independent of reverse transcriptaseinhibition.

Further, the present inventors also tested whether NRTIs blockedinflammasome activation by LPS/ATP, which is not known to signal viareverse transcriptase (Mariathasan et al., 2004; Mariathasan et al.,2006; Martinon et al., 2002). It was found that d4T inhibitedLPS/ATP-induced Caspase-1 maturation in primary mouse bonemarrow-derived macrophages (FIG. 38) as detected by Western blot.

Caspase-1 directly processes interleukin 1 beta (IL-1 beta) upon LPS/ATPstimulation; d4T also blocks secretion of mature IL-1 beta in thesecells (FIG. 39). To determine whether LPS/ATP-induced inflammasomeactivation can be inhibited without RT inhibition, the present inventorsutilized thymidine kinase-deficient (Raji/TK⁻) and -expressing(Raji/TK⁺) cells (Balzarini et al., 1989). After addition of AZT, TK⁺,but not TK⁻ cells, the present inventors produced AZT-triphosphate(AZT-TP), the AZT metabolite required for RT inhibition (FIG. 40; FIG.46, FIG. 47, FIG. 48, FIG. 49, FIG. 50). Even though AZT was notphosphorylated in TK⁻ cells, AZT still inhibited LPS/ATP-inducedinterleukin-1 beta maturation (FIG. 41), indicating that AZT did notinhibit interleukin-1 beta maturation via reverse transcriptaseinhibition.

Alu RNA (Kerur et al., 2013) and LPS/ATP (Qu et al., 2011) activate theinflammasome via the ATP receptor P2X7. The present inventors thereforehypothesized that d4T blocks P2X7 or some P2X7-dependent pathway. First,testing was conducted to determine whether d4T acts upstream of P2X7 bymodulating ATP levels; however, d4T does not block release of ATP tocell culture media induced by Alu RNA (FIG. 42).

Next, testing was conducted to determine whether d4T directlyantagonizes P2X7 function: upon ATP binding, cell-surface P2X7 formsnon-selective cation channels that mediate inflammasome activation(Kahlenberg and Dubyak, 2004; Petrilli et al., 2007). However, d4T didnot significantly modulate P2X7 cation channel function as monitored bypatch clamp analysis of HEK293 cells expressing either the mouse or ratP2X7 receptor (Humphreys et al., 2000).

Finally, P2X7 activation is associated with the formation of a largepore that is permeable to molecules of up to ˜1000 Da (Adinolfi et al.,2005; Cheewatrakoolpong et al., 2005; Surprenant et al., 1996). It wasfound that d4T, and also AZT and 3TC, inhibited P2X7-dependent uptake ofthe fluorescent dye YO-PRO1 (M.W. Da) in human P2X7-overexpressingHEK293 stable cell line (FIG. 43) after addition of the selective P2X7agonist bzATP.

Consistent with the idea that NRTIs block Alu-induced P2X7-mediatedinflammasome activation via a mechanism involving dye uptake, AluRNA-induced Caspase-1 activation was inhibited by a small peptide thatblocks P2X7-mediated dye uptake and LPS/ATP-induced inflammasomeactivation, but not cation flux (Pelegrin and Surprenant, 2006) (FIG.51). On the other hand, Alu-induced Caspase-1 activation was notinhibited by calmidazolium, which selectively blocks P2X7-mediatedcation flux but not dye uptake (FIG. 52).

Furthermore, the intracellular C-terminus of P2X7 governsP2X7-associated dye uptake, and a version of d4T that is not cellpermeable (Agarwal et al., 2011) does not block caspase-1 activation byAlu RNA (FIG. 53, FIG. 32). Consistent with antagonism at or downstreamof P2X7, but upstream of mitochondrial dysfunction, d4T blocksmitochondrial ROS (mtROS) production, which are produced upon LPS/ATPstimulation (Adinolfi et al., 2005; Cruz et al., 2007; Garcia-Marcos etal., 2005; Nakahira et al., 2011) and Alu overexpression (Tarallo etal., 2012) was measured by MitoSOx assay (FIG. 54). Finally, d4T doesnot prevent P2X7-independent interleukin 1-beta secretion in PMA-primedTHP-1 cells treated with crystalline monosodium urate (FIG. 11)(Martinon et al., 2006; Riteau et al., 2012).

To explore the potential therapeutic relevance of NRTIs beyond theAlu-induced model of geographic atrophy (GA), it was hypothesized thatif NRTIs function as generic inflammasome inhibitors, then they might bebroadly useful in other animal models of disease that are also driven byP2X7. In the NLRP3 inflammasome- and P2X7-driven graft-versus-hostdisease model (Jankovic et al., 2013; Wilhelm et al., 2010), treatmentof mice receiving allogeneic bone marrow and T cells with d4T showedimproved survival compared to saline treated controls (30-70% vs. 0%).Furthermore, in the NLRP3- and P2X7-driven model of sterile inflammation(McDonald et al., 2010), d4T reduced neutrophil migration to the focusof liver injury.

Interestingly, it has been shown that P2X7-dependent pore function alonecan influence phenotype (Sorge et al., 2012). However, at present, thereare not any FDA-approved drugs that selectively target downstream P2X7signaling and not ion channel activation. Therefore, NRTIs could bevaluable both clinically and experimentally in the selective targetingof P2X7 function.

A role for P2X7 in regulating HIV replication was recently proposed(Hazleton et al., 2012), and HIV patients have increased plasma IL-18levels (Ahmad et al., 2002; Iannello et al., 2010), which decrease aftertreatment with NRTI-containing highly active anti-retroviral therapy(Stylianou et al., 2003). Notably, reduction of plasma IL-18 levels byNRTI treatment of HIV-1 infected patients did not significantlyassociate with viral load or CD4+ T-cell counts (David et al., 2000),indicating that NRTIs can dampen IL-18 levels before inhibition of viralreplication occurs. IL-18 maturation requires pro-IL18 cleavage byactive Caspase 1, which typically also requires P2X7 activation. Thus,the methods and experiments of the present disclosure are consistentwith the idea that NRTIs can modulate HIV-induced cytokine expressionindependent of reverse transcriptase inhibition.

In some embodiments, d4T prevents RPE degeneration induced by Alu RNA inwild type mice. As shown in FIG. 1, sub-retinal Alu RNA administrationto mice causes RPE degeneration in a mouse model of age-related maculardegeneration. Indeed, as shown, d4T co-delivered to the vitreous humorof wild type mice prevents Alu RNA-induced RPE cell death in adose-dependent manner at one week after delivery. The top row of FIG. 1provides an ocular fundus photograph of mice receiving control PBS, orAlu RNA treatment, with or without increasing amounts of d4T (left toright). Arrows denote depigmented regions of RPE cell death, whichresolve at highest dose of d4T. The bottom row of FIG. 1 shows an RPEflat mount, stained for intercellular junctions (ZO-1) in red that aredisrupted upon Alu RNA administration, but restored to healthy RPEmorphology/intercellular junctions at highest dose of d4T.

Meanwhile, in certain embodiments, d4T protects against cytotoxicityinduced by plasmid expressing Alu RNA in vitro. FIG. 2 shows that human(HeLa) cells treated with an enforced expression plasmid for Alu RNA(pAluA) for denoted amounts of time exhibited profoundly reducedviability compared to a null plasmid (pUC19), as monitored by MTSproliferation assay, and that d4T co-administration prevented cell deathinduced by Alu overexpression.

In some exemplary embodiments, d4T does not rescue cytotoxicity viareduction in Alu RNA levels. As presented in FIG. 3, primary human RPEcells treated with antisense oligonucleotides targeting DICER1 (Dcr as)(lane 3 (third lane from left)) show increased Alu RNA levels in thenuclear compartment compared to control antisense oligonucleotides (Ctras) (lane 1 (leftmost)), monitored by Northern blotting using anAlu-specific probe. Meanwhile, co-administration of d4T (lanes 2 and 4)does not reduce Alu RNA levels. FIG. 3 shows u6 (bottom row) as aloading control for nuclear fraction.

Moreover, in some embodiments, d4T does not reduce Alu RNA levels. Forexample, primary human RPE cells may be transfected with Alu RNA, withor without d4T. (FIG. 4) And, as presented in FIG. 4, co-administrationof d4T does not change Alu RNA levels at 1, 4, or 24 hours aftertransfection in the nuclear fraction, as detected by Northern blottingusing an Alu-specific probe. U6 (bottom row) is shown as loading controlfor nuclear fraction in FIG. 4.

The present disclosure further provides that, in some embodiments, d4Tinhibits inflammasome activation by Alu RNA. Indeed, Alu RNA causesNLRP3 inflammasome activation, which is marked by processing of theenzyme Caspase 1, and FIG. 5 provides a Western blot showing that AluRNA causes Caspase-1 maturation in primary human RPE cells at 24 hoursafter Alu administration (Top, Lane 2, lower band), which is blocked byco-treatment with d4T (100 uM; Lane 3). The bottom row in FIG. 5 is avinculin loading control.

In certain embodiments, 3TC inhibits inflammasome activation by Alu RNA.Indeed, Alu RNA causes NLRP3 inflammasome activation, which is marked byprocessing of the enzyme Caspase 1. FIG. 6 is a Western blot showingthat Alu RNA causes Caspase-1 maturation in primary human RPE cells at24 hours after Alu administration (top, lane 2, lower band), which isblocked with co-treatment with 3TC (20-100 uM; lane 3). On the bottom,the loading control, vinculin, is visible.

Next, FIG. 7 provides evidence of AZT, cordycepin, and abacavirinhibition of inflammasome activation by Alu RNA. Indeed, Alu RNA causesNLRP3 inflammasome activation, which is marked by processing of theenzyme Caspase 1. FIG. 7 is a Western blot showing that Alu RNA causesCaspase-1 maturation in primary human RPE cells at 24 hours after Aluadministration (top, lane 2, lower band), which is blocked withco-treatment with azidothymidine (AZT), cordycepin, and abacavir (ABC)(50-100 uM; Lanes 3-8). Again, the loading control vinculin is shown onthe bottom.

In certain embodiments, the present disclosure provides that d4Tinhibits inflammasome activation by LPS/ATP. As such, FIG. 8 provides agel showing that primary human RPE cells treated with LPS/ATP, a classicinflammasome activator, exhibit increased Casp-1 activation, andphosphorylation of IRAK4, which is also a marker of inflammasomesignaling via the cell surface receptor adaptor protein MyD88. Moreover,as shown in FIG. 8, d4T (25/100 uM) blocks Casp-1 activation and IRAK4phosphorylation induced by LPS/ATP. The loading control in FIG. 8 isvinculin. Furthermore, as shown, LPS and ATP activate the NLRP3inflammasome only in combination, thus treatment with one or the otheralone is useful as a control for this experiment.

The present disclosure further provides that, in exemplary embodiments,d4T and other NRTIs reduce inflammasome activation by LPS/ATP. Aspresented in FIG. 9, d4T, 3TC, and cordycepin (at 100 uM), all di-deoxynucleoside reverse transcriptase inhibitors, inhibit Caspase-1activation (active p20 band, top) and IL-18 maturation (bottom) inducedby LPS/ATP. To produce FIG. 9, cell culture supernatants were collectedafter (i) no treatment, (ii) LPS treatment, or (iii) LPS/ATP treatmentof mouse bone marrow-derived macrophages and run on Western blottingprobing with antibodies for Caspase-1 and IL-18.

In some embodiments of the present disclosure, d4T inhibitsnigericin-induced inflammasome activation. Per FIG. 10, d4T (100, 250uM) inhibits IL-1 beta maturation (top, 18 and 22 kDa forms) andCaspase-1 activation (active p20 band, bottom) induced by nigericin.Cell culture supernatants were collected after (i) no treatment, (ii)LPS treatment, or (iii) LPS/nigericin treatment of mouse bonemarrow-derived macrophages, and run on Western blotting probing withantibodies for IL-1 beta and Caspase-1. FIG. 10 shows the results ofthese efforts.

Additionally, in some embodiments, d4T does not inhibit IL-1 betasecretion from PMA-differentiated THP-1 monocytes induced by MSU. HumanTHP-1 monocytes were differentiated into macrophages with PMA. As shownin FIG. 11, treatment with monosodium urate (MSU), a known inflammasomeactivator, increased IL-1 beta secretion compared to non-treated cells,whereas d4T co-administration at a range of doses (25-1000 uM) did notsignificantly affect IL-1beta secretion. Further, d4T does not blockMSU-induced IL-1 beta secretion as determined by ELISA (n=3-4).

In certain embodiments, d4T and other nucleoside reverse transcriptaseinhibitors do not inhibit IL-1 beta secretion from PMA-differentiatedTHP-1 monocytes induced by MSU. To illustrate this, human THP-1monocytes were differentiated into macrophages with PMA. Treatment withMSU increased IL-1 beta secretion compared to non-treated cells. (FIG.12) Meanwhile d4T, 3TC, or cordycepin (all are di-deoxy nucleotideanalogs) co-administration at a range of doses (25-1000 uM) did notsignificantly affect IL-1beta secretion, as shown in FIG. 12.

Next, in some embodiments, d4T reduces NLRP3 priming induced by Alu RNA.Indeed, as provided in the bar graph of FIG. 13, Alu RNA transfectionincreases NLRP3 mRNA levels in primary human RPE cells at 16 hours, anevent termed “priming” (Y-axis) compared to mock (transfection reagentalone). This effect is blunted by co-administration of d4T (100 uM) andnormalized to 18S RNA control.

Furthermore, in exemplary embodiments of the present disclosure, d4Treduces IL-1beta priming induced by Alu RNA. FIG. 14 illustrates thatAlu RNA transfection increases IL-1 beta mRNA levels in primary humanRPE cells at 24 hours, an event termed “priming”, (Y-axis) compared tomock (transfection reagent alone). This effect is blunted byco-administration of d4T (100 uM) and normalized to 18S RNA control.

Meanwhile, in some embodiments, d4T reduces mitochondrial ROS caused byAlu expression. FIG. 15 demonstrates that enforced expression of Alu(pAluA) causes increased mitochondrial reactive oxygen species (mtROS),as detected by MitoSox assay. In order to produce FIG. 15, primary humanRPE cells were incubated with Alu expressing plasmid or control plasmid(pUC19) with or without d4T. After 15 hours cells were co-stained formtROS (red) and for cell count, nuclei (blue; Hoechst DNA stain). Cellsin the pAluA group exhibited greater mtROS staining (red) compared topUC19 control, an effect that is reduced in pAluA+d4T treated cells.

And in further embodiments, d4T does not inhibit ATP release induced byAlu RNA. (FIG. 16) Primary human RPE cells treated with Alu RNA for thetimes indicated release ATP. To provide FIG. 16, cell culturesupernatant was collected from mock or Alu RNA treated cells, with orwithout d4T. ATP was detected using an ATP-dependent luciferase assay.And, notably, d4T did not affect ATP release.

In certain embodiments, d4T reduces ATP-induced cell permeability toYo-Pro1 (P2X7 receptor assay), as shown in FIG. 17. To prepare FIG. 17,THP-1 cells differentiated into macrophages by PMA allowed entry of thelarge fluorescent dye Yo-Pro 1, in an assay for P2X7 receptor activity.It was observed that d4T dose-dependently reduced Yo-Pro entry inducedby ATP, determined by an area-scan fluorescent measurement in a 96 wellmicroplate reader. Indeed, FIG. 17 provides the results of thefluorescence measurement in relative fluorescence units (RFU, y-axis).

Furthermore, it has been shown that d4T reduces extracellular potassiumlevels that increase after Alu RNA transfection. (FIG. 18) Indeed, cellculture potassium levels increase in primary human RPE cells treatedwith Alu RNA for 6 hours, an effect that is reduced by d4Tco-administration. For FIG. 18, potassium levels were determined in cellculture supernatants spectrophotometrically using a potassium-dependentpyruvate kinase assay.

Next, in some embodiments, d4T blocks bzATP-induced cell permeability toYo-Pro1 (P2X7 receptor assay), as shown in FIG. 19. d4T blocked YO-PRO-1iodide entry in HEK293 cells stably expressing the human P2X7 receptorstimulated with the P2X7-selective agonist bzATP. Cells werepre-incubated with d4T for 30 minutes prior to addition of bzATP/YO-PRO,and fluorescence at 485/515 nm measured at t=30 minutes.

Moreover, d4T blocks Alu-induced RPE degeneration and Caspase-1activation independent of reverse transcriptase inhibition.

In some embodiments, the present disclosure is directed to a compoundhaving the structure(s) provided in FIG. 20. FIG. 20 includes a chemicalstructure of methoxy-d4T (me-d4T) and of d4T. As shown in FIG. 20, asingle substitution of the ribose 5′ hydroxyl group with a methoxy group(circled) has been designed by the inventors of the present disclosureto prevent d4T phosphorylation. Accordingly, in some embodiments, thepresent disclosure is directed to a compound comprising a singlesubstitution of a ribose 5′ hydroxyl group with a methoxy group. And, insome embodiments, the present disclosure provides compounds comprising amethoxy group in place of a ribose 5′ hydroxyl group for preventingphosphorylation, such as d4T phosphorylation.

The present disclosure further provides the results of additionalexperiments in FIG. 21-FIG. 23. Indeed, FIG. 21 is a Western blot ofCaspase-1 activation (p20 subunit) in primary human RPE cellstransfected with Alu RNA±me-d4T; FIG. 22 shows cells, wherein unmodifiedd4T, but not me-d4T, blocks replication of a GFP-expressing lentivirusin HeLa cells; and FIG. 23 provides a graph illustrating that unmodifiedd4T, but not me-d4T, reduces mtDNA levels (normalized to chromosomal DNAexon-intron junction sequence) in primary mouse RPE cells as determinedby real-time quantitative PCR. n=4, *p<0.05 by Student's t-test.

In some embodiments, it has been shown that Me-d4T (intraperitonealinjection) prevents Alu-induced RPE degeneration in mice. FIG. 24, toprow, provides flat mounts stained for zonula occludens-1 (ZO-1; red),bottom row. Degeneration is outlined if FIG. 24 by blue arrowheads.Representative images of n=4 are shown.

Meanwhile, FIG. 25 provides a schematic overview of me-d4T synthesis,and FIG. 26 is an HPLC chromatogram of me-d4T (peak #6) finalproduct, >97% purity. And FIG. 27 is a 1H NMR spectroscopy of me-d4Tfinal product, wherein the chemical shifts are consistent with thestructure, and FIG. 28 provides the results of liquidchromatography/mass spectrometry of me-d4T final product, m/z ratioconsistent with the structure.

FIG. 29, FIG. 30 and FIG. 31 provide for methoxy variants of nucleosideanalogs. Specifically, FIG. 29 shows the chemical structure of 3TC (2′3′dideoxycytidine); FIG. 30 provides the chemical structure of AZT(3′-azido-2′,3′-dideoxythymidine); and FIG. 31 shows the chemicalstructure of ABC (cyclopropylaminopurinylcyclopentene). In each of FIGS.29-31, the methoxy variation (O-methyl group) of nucleoside analog iscircled. Further, FIG. 32 shows a cell permeant variant of d4T (IC-d4T),where “n” group is equal to 11. Derivatives include cell permeantvariants of 3TC, AZT, ABC, where the nucleobase group (circled) may bereplaced, in various embodiments, by 3TC, AZT, ABC, or methoxy-variantsof d4T, 3TC, AZT, ABC (FIG. 29-31), or derivatives thereof.

Meanwhile, FIG. 33 provides the chemical structure of an exemplary NRTIaccording to the present disclosure.

In certain embodiments, the present disclosure provides that NRTIs blockAlu-induced RPE degeneration and/or Caspase-1 activation. For example,FIG. 34 shows a Western blot of Caspase-1 activation (p20 subunit) andIRAK4 phosphorylation in primary human RPE cells transfected with AluRNA±d4T. FIG. 35 is a Western blot of Caspase-1 activation in human RPEcells transfected with Alu RNA±NRTIs (3TC, AZT, ABC). FIG. 36 shows thatpAlu causes RPE degeneration, which is prevented by oral administrationof d4T, and FIG. 37 shows that pAlu causes RPE degeneration, which isprevented by intraperitoneal administration of AZT. FIG. 36 and FIG. 37include fundus photographs: top row; flat mounts stained for zonulaoccludens-1 (ZO-1; red), bottom row. Degeneration is outlined by bluearrowheads. Scale bars, 50 μm.

FIGS. 38-41 illustrate that NRTIs block LPS/ATP-induced inflammasomeactivation. FIGS. 38 and 39 show that d4T blocked Caspase-1 (FIG. 38)and IL-1 beta (FIG. 39) activation in LPS/ATP treated primary mouse bonemarrow-derived macrophages as determined by western blot of cell culturemedia and lysate. Moreover, FIG. 40 presents chromatograms showing thatRaji TK⁺ cells, but not Raji TK⁻ cells, phosphorylate AZT toAZT-triphosphate (AZT-TP) as determined by liquid chromatography-massspectrometry (LC-MS). And FIG. 41 shows that AZT blocks IL-1 betaactivation by LPS/ATP in both Raji TK⁻ and TK⁺cells as determined bywestern blot of cell lysates. Representative images of n=3-4 experimentsare provided in each of FIGS. 38-41.

In some embodiments, the present disclosure provides that NRTIsselectively block P2X7 pore function and P2X7-driven models of graftrejection and sterile liver inflammation, as shown in FIGS. 42-43. FIG.42 is a bar graph illustrating that d4T does not block Alu-induced ATPrelease from primary human RPE cells (n=4). Meanwhile, FIG. 43 is agraph illustration showing that NRTIs selectively block P2X7 porefunction and P2X7-driven models of graft rejection and sterile liverinflammation, providing a graph of the fluorescence (% of bzATP) overtime (minutes).

And in certain exemplary embodiments, the present disclosure providesthat d4T blocks Caspase-1 activation without reducing Alu RNA levels.Accordingly, FIG. 44 provides a Western blot of Caspase-1 activation(p20 subunit) and IRAK4 phosphorylation in primary mouse RPE cellstransfected with Alu RNA±d4T. And FIG. 45 presents a Northern blot ofbiotin-UTP-labeled Alu RNA-transfected primary human RPE cells. Notably,in FIG. 45, d4T did not reduce Alu RNA levels (normalized to u6 RNA).

Next, FIGS. 46-47 provide LC-MS/MS spectra of AZT-triphosphate (AZT-TP,target compound; FIG. 46) and AZU-triphosphate (AZU-TP, internalstandard; FIG. 47). And FIGS. 48-49 show the chromatographic separationof Raji TK⁻ cells spiked with AZT-TP (FIG. 48) and AZU-TP (FIG. 49) withMS spectra (insets) to confirm identity of designated peaks.

FIG. 50 is a standard curve of AZT-TP standards (black circle). Raji TK⁺samples treated with AZT produced AZT-TP (white triangles), whereasAZT-TP was not detectable in Raji TK⁻ cells treated with AZT. FIG. 50 isrepresentative of two experiments.

FIGS. 51-54 show that, in some exemplary embodiments, P2X7-dependentpore function mediates Alu-induced Caspase-1 activation. Indeed, FIG. 51is a Western blot of Caspase-1 activation (p20 subunit) in primary humanRPE cells transfected with Alu RNA, with short peptide (Panx1¹⁰), whichblocks P2X7 pore function but not cation flux (vs. scrambled peptide:Scr Panx1¹⁰); FIG. 52 is a Western blot of Caspase-1 activation (p20subunit) in primary human RPE cells transfected with Alu RNA, withcalmidazolium (FIG. 32 provides the chemical structure of IC- and EC-d4Tused), which blocks P2X7 cation flux but not pore function; and FIG. 53is a Western blot of Caspase-1 activation (p20 subunit) in primary humanRPE cells transfected with Alu RNA, with cell permeable (IC),cell-impermeable (EC), or unmodified (no tag) d4T. Furthermore, FIG. 54shows that d4T prevents pAlu-induced mitochondrial ROS generation inprimary human RPE cells. In FIG. 54, mitochondrial reactive oxygenspecies (ROS) were visualized with MitoSox (Red) and cell nuclei withHoechst (Blue).

Caspase-1 activation (p20 subunit) in primary human retinal pigmentepithelium (RPE) cells in the presence of iron (III) ammonium citratewas studied. The addition of TM-3TC (Structure 8) (25 μM) blockediron-induced Caspase-1 activation, as reflected in the western blot setforth in FIG. 55. The addition of 2me-d4T (Structure 4) (25 μM) blockediron-induced Caspase-1 activation, as reflected in the western blot setforth in FIG. 57.

Caspase-1 activation (p10 subunit) in primary human RPE cells in thepresence of iron (III) ammonium citrate was studied. The addition ofd4T-ene (Structure 3) (25 μM) blocked iron-induced Caspase-1 activation,as reflected in the western blot set forth in FIG. 56.

Caspase-1 activation (p20 subunit) in primary human RPE cellstransfected with Alu RNA. The addition of d4T-ene (Structure 3) andTM-3TC (Structure 8) (25 and 100 μM) blocked Alu-induced Caspase-1activation, as reflected in the western blot set forth in FIG. 58.

The relative quantity of mtDNA in primary human RPE cells treated withNRTIs or derivatives (50 μm for each) was studied using vehicletreatment (DMSO) (“No Tx”) as a control. DNA was collected after fourdays in cell culture with exposure to drug. Quantitative polymerasechain reaction was performed for mtDNA and normalized to genomic DNAsequence. As reflected in FIG. 59, modified versions of d4T that are notphosphorylated (e.g. me-d4T (FIG. 20), 2 me-d4T (Structure 4)) do notexhibit mtDNA depletion compared to parental NRTI (d4T). TM-3TC isstructure 8. As reflected in FIG. 60, modified versions of AZT that arenot phosphorylated (me-AZT (Structure 1), N-Me-Me-AZT (Structure 10) donot exhibit mtDNA depletion compared to parental compound (AZT).

Mouse studies were conducted to study treatment with NRTIs orderivatives. Mice receiving subretinal control empty-vector plasmid(pNull), or Alu RNA-expressing plasmid (pAlu), were treated with twicedaily intraperitoneal modified NRTIs (me-AZT (Structure 1), N-me-me-AZT(Structure 10), or 2 me-d4T (Structure 4); 25 mg/kg/administration) orcontrol vehicle. The top row of FIG. 61 displays ocular fundusphotographs of the mice. The bottom row of FIG. 61 displays RPE flatmounts, stained for intercellular junctions (ZO-1) in red that aredisrupted upon Alu RNA expression but that are restored to healthy RPEmorphology/intercellular junctions with modified NRTI treatment.

A schematic overview of the synthesis of Formula I (structure C inScheme 1), Formula IV (structure B), Formula VIII (structure E), FormulaX (structure D), and methoxy-d4T (structure A; also FIG. 20) is setforth in FIG. 62. The general synthesis procedure is as follows: To asuspension of nucleotide (1.5 mmole) in dry THF (5 mL)) was added NaH(15 mmole) and the mixture was stirred for 10 minutes at roomtemperature under nitrogen. Methyl Iodide (15 mmole) was added in oneportion to the mixture and stirred for 1-3 h. The reaction was checkedfor completion by TLC and quenched by drop-wise addition of methanol.The mixture was neutralized with acetic acid and evaporated. The residuewas suspended in dichloromethane and washed with aqueous NaHSO3solution, dried over MgSO4 and evaporated the solvent. The product waspurified by flash column chromatography using silica gel and 2% methanolin dichloromethane. The structures of derivatives were confirmed by LCMSand 1H-NMR spectroscopy.

Structure A) yield 200 mg (56%). 1H NMR (500 MHz, DMSO) 11.31 (s, 1H,NH), 7.50 (d, 1H, 6-H), 6.82 (dd, 1H, I′-H), 6.42 (dd, 1H, 3′-H), 5.91(dd, 1H, 2′-H), 4.88 (s, 1H, 4′-H), 3.56 (m, 2H, 5′-H), 3.28 (s, 3H,OCH3), 1.75 (s, 3H, CH3).

Structure B) 67 mg (18.7%). 1H NMR (500 MHz, DMSO) 7.56(s, 1H, 6-H),6.88 (dt, 1H, 1′-H), 6.43 (dd, 1H, 3′-H), 5.90 (d, 1H, 2′-H), 4.89 (s,1H, 4′-H), 3.56 (m, 2H, 5′-H), 3.27 (s, 3H, OCH3), 3.18 (s, 3H, NCH3),1.8 (s, 3H, CH3).

Structure C) 1 g-scale yield was 0.4 g solid (36%).1H NMR(500 MHz, DMSO)11.31 (s, 1H, NH), 7.56 (d,1H, 6-H), 6.88 (dt, 1H, 1′-H), 6.43 (dd, 2H,3′-H), 5.91 (m, 2H, 2′-H), 4.89(s, 1H, 4′H), 6.43 (dd, 1H, 3′-H), 5.91(d, 2H, 2′-H), 4.89 (s, 1H, 4′-H), 3.55(t, 2H, 5′-H), 3.27 (s, 3H,OCH3), 3.18 (s, 3H, NCH3), 1.8 (d, 3H, CH3).

Structure D) oil (0.5 g, 46%). 1H NMR (500 MHz, DMSO) 7.56 (d, 1H, 6-H),6.88 (dt, 1H, 1′-H), 6.43 (dd, 2H, 3′-H), 5.91 (m, 2H, 2′-H), 4.89(s,1H, 4′H), 6.43 (dd, 1H, 3′-H), 5.91 (d, 2H, 2′-H), 4.89 (s, 1H, 4′-H),3.55(t, 2H, 5′-H), 3.27 (s, 3H, OCH3), 3.18 (s, 3H, NCH3), 1.8 (d, 3H,CH3).

Structure E) Reaction was complete in one hour. Yield 0.224 g(55%). 1HNMR (500 MHz, DMSO) 7.81 (d, 1H, H6), 6.23 (t, 1H, H4′), 6.09 (d. 1H,H5), 5.31 (t, 1H, H2′), 3.70 (m, 2H, H6′), 3.43 (dd, 1H, H5′b), 3.34 (s,3H, OCH3), 3.08 (dd, 1H, H5′a), 3.04 (s, 6H, N(CH3)2).

Example 2

In this Example, structures of unique NRTIs, also referred to as“Kamuvudines”, are studied. A procedure for the synthesis of theseKamuvudines is described herein. Also provided is NMR and massspectrometry data for these Kamuvudines and data regarding thebiological activity of Kamuvudines.

Synthesis of 5′-O-Alkyl Substituted and of Di-Alkyl SubstitutedKamuvudines

To a suspension of nucleotide (1.5 m-mole) in dry THF (5 mL) was addedNaH (4.5 m-mole) and the mixture was stirred for 10 minutes at roomtemperature under nitrogen. Alkyl Iodide (4.5 m-mole) was added in oneportion to the mixture and stirred for 1-3 h. The reaction was checkedfor completion by TLC and quenched by drop-wise addition of methanol.The mixture was neutralized with acetic acid and evaporated. The residuewas suspended in dichloromethane and washed with aqueous NaHSO₃solution, dried over MgSO₄ and the solvent was evaporated. The productwas purified by flash column chromatography using silica gel using ethylacetate/hexane as solvent. The structures of derivatives were confirmedby LCMS and ¹H-NMR spectroscopy.

Synthesis of Asymmetric Di-Substituted Kamuvudines.

A) Synthesis of N-substituted nucleosides. To a suspension of nucleotide(1.5 m-mole) in dichloromethane (5 mL) was added NaH (1.5 m-mole) andthe mixture was stirred for 10 minutes at room temperature undernitrogen. Alkyl Iodide (1.5 m-mole) was added in one portion to themixture and stirred for 1-3 h. The reaction was checked for completionby TLC. Wash the extract with brine and dry with anhydrous sodiumsulfate. Evaporate solvent and purify the N-substituted product by flashchromatography.

B) To a suspension of N-substituted nucleotide (1.5 m-mole) indichloromethane (5 mL)) was added NaH (1.5 m-mole) and the mixture wasstirred for 10 minutes at room temperature under nitrogen. Alkyl Iodide(1.5 m-mole) was added in one portion to the mixture and stirred for 1-3h. The reaction was checked for completion by TLC. Wash the extract withbrine and dry with anhydrous sodium sulfate. Evaporate solvent andpurify the di-substituted product by flash chromatography.

NMR and Mass Spectrometry Data for Kamuvudines

1. a) O-Me-d4T

¹H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 7.26 (q, J=1.3 Hz, 1H), 6.85(dt, J=3.6, 1.7 Hz, 1H), 6.44 (dt, J=6.0, 1.6 Hz, 1H), 6.05 (dt, J=6.1,1.8 Hz, 1H), 5.04 (s, 1H), 4.41 (t, J=2.7 Hz, 2H), 3.17 (d, J=1.2 Hz,3H), 1.75 (d, J=1.3 Hz, 3H). MS (ESI): [M+Na]⁺ Mass calculatedC₁H₁₄N₂O₄Na⁺=261.23, found=261.2.

1. b) 2-Me-d4T

¹H NMR (400 MHz, DMSO-d6) δ 7.56 (q, J=1.2 Hz, 1H), 6.89 (ddd, J=3.4,1.9, 1.4 Hz, 1H), 6.44 (dt, J=6.0, 1.8 Hz, 1H), 5.91 (ddd, J=6.0, 2.5,1.4 Hz, 1H), 4.96-4.83 (m, 1H), 3.60-3.52 (m, 2H), 3.28 (s, 3H), 3.18(s, 3H), 2.08 (s, 2H), 1.81 (d, J=1.2 Hz, 3H). MS (ESI): [M+Na]⁺ Masscalculated C₁₂H₁₆N₂O₄Na⁺=275.28, found 275.2.

1. c) 2-Et-d4T

¹H NMR (400 MHz, DMSO-d6) δ 7.50 (t, J=1.3 Hz, 1H), 6.89 (dq, J=3.4, 1.5Hz, 1H), 6.44 (dt, J=5.9, 1.6 Hz, 1H), 5.93 (ddt, J=6.0, 2.5, 1.4 Hz,1H), 4.90 (d, J=3.6 Hz, 1H), 3.93-3.78 (m, 2H), 3.59 (td, J=3.1, 1.3 Hz,2H), 3.45 (qt, J=7.0, 1.5 Hz, 3H), 1.80 (d, J=1.3 Hz, 3H), 1.10 (tdd,J=7.0, 5.2, 1.3 Hz, 6H). MS (ESI): [M+Na]⁺ Mass calculatedC₁₄H₂₀N₂O₄Na⁺=303.3, found 303.2

1. d) N-Et d4T

¹H NMR (400 MHz, DMSO-d6) δ 7.69 (q, J=1.2 Hz, 1H), 6.89 (dt, J=3.4, 1.7Hz, 1H), 6.41 (dt, J=6.0, 1.8 Hz, 1H), 5.92 (ddd, J=6.0, 2.4, 1.4 Hz,1H), 5.01 (t, J=5.3 Hz, 1H), 4.84-4.75 (m, 1H), 3.85 (qd, J=7.1, 1.8 Hz,2H), 3.60 (dd, J=5.3, 3.4 Hz, 2H), 1.78 (d, J=1.2 Hz, 3H), 1.09 (t,J=7.0 Hz, 3H). MS (ESI): [M+Na]⁺ Mass calculated C₁₂H₁₆N₂O₄Na⁺=275.28,found 275.2

1. e) O-Me N-Et d4T

¹H NMR (400 MHz, DMSO-d6) δ 7.55 (d, J=1.3 Hz, 1H), 6.90(dt, J=3.3, 1.7Hz, 1H), 6.45 (dt, J=6.0, 1.7 Hz, 1H), 5.93 (ddd, J=6.1, 2.3, 1.4 Hz,1H), 4.91 (d, J=4.1 Hz, 1H), 3.91-3.81 (m,2H), 3.56 (dd, J=3.1, 1.5 Hz,2H), 3.28 (s, 3H), 1.81 (d, J=1.2Hz, 3H), 1.10 (t, J=7.0 Hz, 3H). MS(ESI): [M+Na]⁺ Mass calculated C₁₃H₁₈N₂O₄Na⁺ 289.31, found 289.2.

2. a) O-MeAZT

¹H NMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H), 7.50 (q, J=1.1 Hz, 1H), 6.82(dt, J=3.3, 1.6 Hz, 1H), 6.41 (dt, J=6.1, 1.7 Hz, 1H), 5.91 (ddd, J=6.2,2.5, 1.3 Hz, 1H), 4.93-4.77 (m, 1H), 3.55 (d, J=3.1 Hz, 2H), 3.28 (s,3H), 1.75(d, J=1.3 Hz, 3H). MS (ESI): [M+H]⁺, C₁₁H₁₅N₅O₄ ⁺, calculated282.26, found 282.2

2. b) 2-Me-AZT

¹H NMR (400 MHz, DMSO-d6) δ 7.63 (q, J=1.2 Hz, 1H, H6), 6.15 (t, J=6.4Hz, 1H, H1′), 4.43 (dt, J=7.3, 5.4 Hz, 1H, H3′), 3.97 (dt, J=5.1, 4.1Hz,1H, H4′), 3.67-3.50 (m, 2H, H5′), 3.35 (s, 3H, OCH₃), 3.17 (s, 3H,NCH₃), 2.46-2.27 (m, 2H, H2′), 1.85 (d, J=1.2 Hz, 3H, CH3).

MS (ESI): [M+H]⁺ C₁₂H₁₈N₅O₄ ⁺, calculated=296.29, found 296.2

2. c) 2-Et-AZT

¹H NMR (400 MHz, DMSO-d6) δ 7.62 (t, J=1.2 Hz, 1H, H6),6.16 (t, J=6.4Hz, 1H, H1′), 4.43 (q, J=5.8 Hz, 1H, H3′), 3.96 (q, J=4.4 Hz, 1H, H4′),3.83 (q, J=7.0 Hz, 2H), 3.68-3.55 (m, 2H, H5′),3.55-3.44 (m, 2H), 2.37(dp, J=20.5, 7.0 Hz, 2H, H2′), 1.84 (d, J=1.2 Hz, 3H, CH₃), 1.15 (td,J=7.0, 1.1 Hz, 3H), 1.08 (t, J=7.0 Hz, 3H).

MS (ESI): [M+H]⁺ calculated C₁₄H₂₂N₅O₄ ⁺=324.35, found 324.2.

3. a) 3-Me-3TC

¹H NMR (400 MHz, DMSO-d6) δ7.82 (d, J=7.8 Hz,1H), 6.23 (t, J=5.2 Hz,1H), 6.09 (d, J=7.8 Hz, 1H), 5.31(t, J=4.7 Hz, 1H), 3.77-3.65 (m, 2H),3.44 (dd, J=11.7,5.5 Hz, 1H), 3.34 (s, 3H), 3.09 (dd, J=11.7, 4.9 Hz,1H), 3.05 (s, 6H).

MS (ESI): [M+H]⁺, C₁₁H₁₈N₃O₃S⁺ calculated 272.34, found 272.2

3. b) 3-Et-3TC

¹H NMR (400 MHz, DMSO-d6) δ 7.89-7.77 (m, 1H), 6.22 (d, J=4.7 Hz, 1H),6.03 (d, J=8.2 Hz, 1H), 5.29 (d, J=4.6 Hz, 1H), 3.74 (d, J=4.7 Hz, 2H),3.60-3.48 (m,4H, CH₂), 3.32 (s, 2H), 3.10 (d, J=12.1 Hz, 1H),1.18-1.01(m, 9H, CH₃).MS (ESI): [M+H]⁺, C₁₄H₂₄N₃O₃S⁺ calculated 314.41,found 314.4

Characteristics of NRTIs and Kamuvudines

In some embodiments, as compared to the original NRTIs (d4T, 3TC, AZT),the Kamuvudines (modified NRTIs) have more desirable drug-likecharacteristics. For example, referring to Table 1 below, as compared tothe NRTIs, the Kamuvudines have greater LogP values (greater than 0 andclose to 1) and lower solubility in water. During certain types ofcompound release, such as in an intraocular sustained release drugdelivery system, the greater LogP values and lower solubility of theKamuvudines provide greater resident times (i.e., longer half-lives) inthe vitreous humor and retina, as compared to the original NRTIs.

TABLE 1 Name LogP Solubility (mg/ml) d4T −0.23 106 Me-d4T 0.41 302Me-d4T 0.63 10 2Et-d4T 1.35 0.09 O—Me, N—Et-d4T 0.99 1.5 3TC −1.1 64TM-3TC 0.09 7 3Et-3TC 1.16 1.6 AZT −0.41 16 2Me-AZT 0.45 0.94 2Et-AZT1.17 0.01 LogP: Partition coefficient in 1-octanol. Solubility in WaterpH 7.2 at 37° C.

Efficacy of NRTIs and Kamuvudines in Cells

With reference to FIGS. 64-73, and 74-78, compounds as disclosed hereininhibit Caspase-1 activation. Caspase-1 is the enzyme at the core of theinflammasome complex and is an important danger response signal andmediator of RPE cell death in AMD. FIGS. 64-73 show that Kamuvudinesdose-dependently inhibit Caspase-1 as determined by the iGlucluminescence assay in mouse J774 iGluc cells. Importantly, there wassome difference between individual compounds in terms of the doseresponse curve, emphasizing the need to test each compound (with uniqueR group(s)) individually. Furthermore, Kamuvudines exhibited variable,incomplete inhibition of Caspase-1 activation, also suggesting thatthere are differences of compound activity within the class ofKamuvudines.

To obtain the data set forth in FIGS. 64-73, an iGLuc assay wasconducted (modified from Bartok et al. iGLuc: a luciferase-basedinflammasome and protease activity reporter. Nature Methods 2013;10(2):147-54). Briefly, 100,000 iGLuc cells per well were plated in a 96well plate overnight. The next morning, media is aspirated from theplate and replaced with 75 μL of serum free DMEM with drugs (NRTIs ormodified NRTIs) for 30 min. After 30 minutes of drug exposure, 20 mM ATP(25 4) is added for a final concentration of 5 mM. 50 μL of media fromeach well is collected and 50 μL of coelenterazine 4.4 μM is added toeach sample, and luminescence is read immediately.

FIG. 64 shows that 3TC inhibits Caspase-1 activation. ATP (5 mM) inducesCaspase-1 activation, which is measured by luminescence monitoring ofLuciferase cleavage by Caspase-1 in mouse J774 iGLuc cells. Exposure to3TC (1 nM-100 μM) reduces Caspase-1 cleavage in a dose-dependentfashion. N=3.

FIG. 65 shows that 3Me-3TC inhibits Caspase-1 activation. ATP (5 mM)induces Caspase-1 activation, which is measured by luminescencemonitoring of Luciferase cleavage by Caspase-1 in mouse J774 iGLuccells. Exposure to 3Me-3TC (1 nM-100 μM) reduces Caspase-1 cleavage in adose-dependent fashion. N=3.

FIG. 66 shows that 3Et-3TC inhibits Caspase-1 activation. ATP (5 mM)induces Caspase-1 activation, which is measured by luminescencemonitoring of Luciferase cleavage by Caspase-1 in mouse J774 iGLuccells. Exposure to 3Et-3TC (1 nM-100 μM) reduces Caspase-1 cleavage in adose-dependent fashion. N=3.

FIG. 67 shows that AZT inhibits Caspase-1 activation. ATP (5 mM) inducesCaspase-1 activation, which is measured by luminescence monitoring ofLuciferase cleavage by Caspase-1 in mouse J774 iGLuc cells. Exposure toAZT (1 nM-100 μM) reduces Caspase-1 cleavage in a dose-dependentfashion. N=3.

FIG. 68 shows that 2Me-AZT inhibits Caspase-1 activation. ATP (5 mM)induces Caspase-1 activation, which is measured by luminescencemonitoring of Luciferase cleavage by Caspase-1 in mouse J774 iGLuccells. Exposure to 2Me-AZT (1 nM-100 μM) reduces Caspase-1 cleavage in adose-dependent fashion. N=3.

FIG. 69 shows that 2Et-AZT inhibits Caspase-1 activation. ATP (5 mM)induces Caspase-1 activation, which is measured by luminescencemonitoring of Luciferase cleavage by Caspase-1 in mouse J774 iGLuccells. Exposure to 2Et-AZT (1 nM-100 μM) reduces Caspase-1 cleavage in adose-dependent fashion. N=3.

FIG. 70 shows that d4T inhibits Caspase-1 activation. ATP (5 mM) inducesCaspase-1 activation, which is measured by luminescence monitoring ofLuciferase cleavage by Caspase-1 in mouse J774 iGLuc cells. Exposure tod4T (1 nM-100 μM) reduces Caspase-1 cleavage in a dose-dependentfashion. N=3.

FIG. 71 shows that O-Me N-Et d4T inhibits Caspase-1 activation. ATP (5mM) induces Caspase-1 activation, which is measured by luminescencemonitoring of Luciferase cleavage by Caspase-1 in mouse J774 iGLuccells. Exposure to O-Me N-Et d4T (1 nM-100 μM) reduces Caspase-1cleavage in a dose-dependent fashion. N=3.

FIG. 72 shows that Me-d4T inhibits Caspase-1 activation. ATP (5 mM)induces Caspase-1 activation, which is measured by luminescencemonitoring of Luciferase cleavage by Caspase-1 in mouse J774 iGLuccells. Exposure to Me-d4T (100 μM) reduces Caspase-1 cleavage in adose-dependent fashion. N=3.

FIG. 73 shows that 2Et-d4T inhibits Caspase-1 activation. ATP (5 mM)induces Caspase-1 activation, which is measured by luminescencemonitoring of Luciferase cleavage by Caspase-1 in mouse J774 iGLuccells. Exposure to 2Et-d4T (1 nM-100 μM) reduces Caspase-1 cleavage in adose-dependent fashion. N=3.

As illustrated in FIGS. 70-73, compared to d4T, there was a greaterreduction in inflammasome activation by the Kamuvudines (modifiedNRTIs). Specifically, d4T reduced inflammasome activation by about 35%,while O-Me, N-Et-d4T reduced inflammasome activation by about 55%,Me-d4T reduced inflammasome activation by about 70%, and 2Et-d4T reducedinflammasome activation by about 45%. Without wishing to be bound bytheory, as each of the Kamuvudines provided a greater reduction ininflammasome activation as compared to the parent NRTI (i.e., d4T), itis believed that the Kamuvudines also provide increased blocking ofretinal degeneration.

FIG. 74 shows that Kamuvudines block Caspase-1 activation induced by AluRNA in primary human RPE cells, as monitored by western blotting. AluRNA induced Caspase-1 activation in primary human RPE cells, monitoredby western blotting, is reduced by 2Et-d4T (25 μM), 2Me-AZT (25 μM),O-Me N-Et d4T (25 μM), d4T-ene (25-100 μM), and TM-3TC (25-100 μM).N=3-4.

Alu RNA is a toxic endogenous retroelement that accumulates in andcauses death of the RPE in patients with dry AMD. FIG. 75, shows thatKamuvudines block Caspase-1 activation in primary human cells exposed toiron (Fe 3+) ammonium citrate (FAC). FAC activates Caspase-1 via an AluRNA intermediate, and induces NLRP3-dependent RPE degeneration (Gelfandet al. Cell Reports 2015). Fe(III) ammonium citrate (100 μM)-inducedCaspase-1 activation in primary human RPE cells, monitored by westernblotting, is reduced by TM-3TC (25 μM), d4T-ene (25 μM), 2Me-d4T (25μM), Me-AZT (25-100 μM), and 2Me-AZT (25 μM). N=3-4.

FIGS. 76-78, show that the NRTI 3TC blocks Caspase-1 activation inducedby AMD-associated stressors (Complement protein C3a, ROS-generatorparaquat, and amyloid beta) in primary human RPE cells. FIG. 76 showsthat NRTIs reduce Complement-induced Caspase-1 activation in human RPEcells. Human C3a (100 nM)-induced Caspase-1 activation after LPS primingin primary human RPE cells, monitored by western blotting, is reduced by3TC (25 μM). N=3-4. FIG. 77 shows that NRTIs reduce Paraquat-inducedCaspase-1 activation in human RPE cells. Paraquat (250 μM)-inducedCaspase-1 activation in primary human RPE cells, monitored by westernblotting, is reduced by 3TC (25 μM). N=3-4. FIG. 78 shows that NRTIsreduce Amyloid beta-induced Caspase-1 activation in human RPE cells.Oligomerize Aβ1-40 peptide (0.5 μM)-induced Caspase-1 activation inprimary human RPE cells, monitored by western blotting, is reduced by3TC (10-25 μM). N=3-4.

Efficacy of NRTIs and Kamuvudines in Mice

The efficacy of Kamuvudines in mouse models of dry and wet AMD is shownherein. With reference to FIGS. 79-87, compounds as disclosed hereinreduce choroidal neovascularization (CNV) in a model of “wet AMD” andblock RPE degeneration in a model of “dry AMD.”

FIGS. 79 and 80 show that NRTIs and Kamuvudines are effective in thelaser-induced model of choroidal neovascularization (CNV; wet AMD). FIG.79 shows that compounds disclosed herein reduce choroidalneovascularization in a model of “wet AMD”. Intravitreous administrationof 3TC (0.55 nmol), ABC (0.64 nmol), or AZT (0.55 nmol) after laserinjury reduced laser injury-induced CNV volume in wild-type mice.Injection of PBS or DMSO were vehicle controls. N=16. FIG. 80 shows thatcompounds disclosed herein reduce choroidal neovascularization (CNV) ina model of “wet AMD”. Intravitreous administration of Me-d4T,O-Me-N-Et-d4T, 2Et-AZT or 3Et-3TC (0.14 nmol) after laser injury reducedchoroidal neovascularization (CNV) volume in wild-type mice at day 7.Injection of DMSO was vehicle controls. N=16. Compared to NRTIs (FIG.79), modified NRTIs (FIG. 80) are more potent (smaller dose required) atsuppressing CNV and are more effective (greater degree of suppressionachieved).

Without wishing to be bound by theory, it is believed that both theNRTIs and the Kamuvudines provide antiangiogenic effects in aP2X7-dependent manner, which reduces CNV. Specifically, intravitreousinjection of the NRTIs or the Kamuvudines suppressed laser-induced CNVin wild-type mice as compared to PBS or DMSO, respectively, whileintravitreous injection of the NRTIs did not suppress the laser-inducedCNV in P2rx7^(−/−) mice. Additionally, intravitreous injection of theNRTIs suppressed laser-induced CNV in Nfrp3^(−/−) mice, indicating thatthe antiangiogenic effects are N/rp3-independent. The P2X7 inhibitionand/or angio-inhibitory effects of the NRTIs and Kamuvudines is alsobelieved to be effective in treating other diseases, including, but notlimited to, blocking tumor growth and/or treating graft-versus-hostdisease.

FIGS. 81 and 82 show that, compared to original NRTIs, Kamuvudines haveincreased effectiveness and potency in treating existing neovasculardisease. JR5558 mice develop spontaneous choroidal neovascularization(CNV) and are an animal model of wet/neovascular age-related maculardegeneration (AMD). The activity of the CNV lesions can be monitoredusing fluorescein angiography to assess the degree of dye leakage fromthe lesions, as is done in humans with this disease. FIGS. 81 and 82 arerepresentative images from two JR5558 mice (N=8). The first and thirdrow of FIGS. 81 and 82 show time dependent dye leakage from pre-existingCNV lesions in the left eye (first row) and right eye (third row) of afirst and second JR5558 mouse, respectively. The second row of FIGS. 81and 82 show time dependent dye leakage in the left eye after a singleintravitreous administration of 125 ng of 3TC, and the fourth row showstime dependent dye leakage in the right eye after a single intravitreousadministration of 62.5 ng of 3-Me-3TC. As seen in rows two and four,both 3TC and 3-Me-3TC suppress the activity of pre-existing CNV lesions3 days after administration (i.e., reduce their dye leakage).Additionally, compared to the original NRTI (3TC), the Kamuvudine (i.e.,3-Me-3TC) provides a greater reduction in CNV lesion leakage (i.e., ismore effective) at lower doses (i.e., is more potent).

In models of dry AMD, in FIGS. 83-86, Kamuvudines are shown to blockAlu-induced RPE degeneration, as monitored by fundus photography andZO-1 flat mount staining of RPE, in a model of dry AMD.

FIG. 83 shows that compounds disclosed herein block Alu RNA-induced RPEdegeneration in a model of “dry AMD”. Subretinal injection of Alu RNA(0.3 μg) induces RPE degeneration (whitish yellow region on color fundusphotograph (top row) and disorganization of hexagonal array (bottom row)in wild-type mice. Intraperitoneal administration of 3Me-3TC (25 mg/kg,twice daily for 6 days after Alu RNA injection) protects against RPEdegeneration, as seen on fundus photos (Top; smaller area of whitisharea of discoloration) and ZO-1 immunostained RPE flat mounts (Bottom;more hexagonal, tessellated appearance). Bottom panels show fundusphotographs that demonstrate that intraperitoneal administration of O-MeN-Et d4T (25 mg/kg, twice daily for 6 days after Alu RNA injection)protects against Alu RNA-induced RPE degeneration in wild-type miceImage representative of 6 experiments.

FIG. 84 shows that 3Me-3TC disclosed herein block pAlu-induced RPEdegeneration in a model of “dry AMD”. Subretinal injection of a plasmidencoding Alu (pAlu) induces RPE degeneration (whitish yellow region oncolor fundus photograph (top row) and disorganization of hexagonal array(bottom row) in wild-type mice. Intraperitoneal administration of3Me-3TC (25 mg/kg, twice daily for 6 days after pAlu injection) protectsagainst RPE degeneration, as seen on fundus photos (Top) and ZO-1immunostained RPE flat mounts (Bottom). Image representative of 6experiments.

FIG. 85 shows that 3Et-3TC blocks pAlu-induced RPE degeneration in amodel of “dry AMD”. Subretinal injection of a plasmid encoding Alu(pAlu) induces RPE degeneration (whitish yellow region on color fundusphotograph (top row) and disorganization of hexagonal array (bottom row)in wild-type mice. Intraperitoneal administration of 3Et-3TC (25 mg/kg,twice daily for 6 days after pAlu injection) protects against RPEdegeneration, as seen on fundus photos (Top) and ZO-1 immunostained RPEflat mounts (Bottom). Image representative of 6 experiments.

FIG. 86 shows that compounds disclosed herein block pAlu-induced RPEdegeneration in a model of “dry AMD”. Subretinal injection of a plasmidencoding Alu (pAlu) induces RPE degeneration (whitish yellow region oncolor fundus photograph (top row) and disorganization of hexagonal array(bottom row) in wild-type mice. Intraperitoneal administration of2Et-d4T or 2Et-AZT (25 mg/kg, twice daily for 6 days after pAluinjection) protects against RPE degeneration, as seen on fundus photos.Image representative of 6 experiments.

In FIGS. 87-89, Kamuvudines are shown to be effective in blocking RPEdegeneration after intraocular injection of poly I:C, amyloid beta, andiron.

FIG. 87 shows that compounds disclosed herein block poly IC-induced RPEdegeneration in a model of “dry AMD”. Poly IC (0.2 μg) administered intothe subretinal space of wild-type mice induces RPE degeneration (whitishyellow region on color fundus photograph (top row) and disorganizationof hexagonal array (bottom row). Intravitreous administration of Me-d4Tor 2-Me-d4T (0.14 mmol) (after poly IC injection) blocks thisdegeneration (measured 7 days after poly IC). Top row showsrepresentative fundus photographs. Bottom row shows representative ZO-1immunostained RPE flat mounts. N=4-6.

FIG. 88 shows that compounds disclosed herein block Amyloid beta-inducedRPE degeneration in a model of “dry AMD”. Oligomerized Amyloid beta 1-40peptide (0.83 μmol) was injected into the subretinal space of mice toinduce RPE degeneration degeneration (whitish yellow region on colorfundus photograph (top row) and disorganization of hexagonal array(bottom row). Intraperitoneal administration of 3TC or K-2 (2Me-d4T) ondays 1 to 6 (25 mg/kg) blocked the development of RPE degeneration onday 7. Top row shows color fundus photographs. Bottom row show ZO-1stained RPE flat mounts.

FIG. 89 shows that compounds disclosed herein block Iron-induced RPEdegeneration in a model of “dry AMD”. Fe(III) ammonium citrate (3 nM)was injected into the subretinal space of mice to induce RPEdegeneration (whitish yellow region on color fundus photograph (top row)and disorganization of hexagonal array (bottom row). Intraperitonealadministration of d4T, K-1 (Me-d4T), or K-2 (2Me-d4T) on days 1 to 6 (25mg/kg) blocked the development of RPE degeneration on day 7 compared tovehicle treatment. Top row shows color fundus photographs. Bottom rowshow ZO-1 stained RPE flat mounts.

Safety/Toxicity of NRTIs vs Kamuvudines in Mice and Cells

Safety and toxicity of the compounds was also studied. FIG. 90 showsthat NRTIs are toxic to the retina after intravitreous injection.Surprisingly and unexpectedly, adding a single R substitution to NRTIseliminated this toxicity: FIG. 91 shows that intravitreous injection ofthe same dose of Kamuvudines is not toxic, indicating that they aresafer than NRTIs for intraocular administration. Furthermore, FIG. 92 weshows that Kamuvudines are safe in that their intravitreousadministration does not induce anatomical disruption of the retina.

FIG. 90 shows that known NRTIs can cause retinal toxicity. Intravitreousinjection of d4T, AZT, or 3TC (0.56 nmol) in mice induced areas ofretinal degeneration (red arrowheads) retinal toxicity. Representativecolor fundus photograph images shown (N=8). These data indicate that,NRTIs could have adverse effects following intraocular administration.

FIG. 91 shows that compounds disclosed herein are not toxic to theretina. Intravitreous injection of Kamuvudines 1-9 (2.8 nmol) in micedid not induce retinal toxicity. Representative color fundus photographimages shown (N=8). These data indicate that, unlike NRTIs, Kamuvudinesare safer for intraocular administration.

FIG. 92 also shows that compounds disclosed herein are not toxic to theretina. Intravitreous injection of Kamuvudines 1-5 (2.8 nmol) in micedid not induce any anatomical disruption of the retina (H&E staining—toprow) or changes in thickness of the various layers of the retina (ILM:internal limiting membrane; OLM: outer limiting membrane; IPL: innerplexiform layer; INL: inner nuclear layer; ONL: outer nuclear layer; IS:inner segments; OS: outer segments). N=8. These data indicate that,unlike NRTIs, Kamuvudines are safer for intraocular administration evenat very high concentrations.

Finally, FIGS. 93 and 94 show that Kamuvudines, lack “off-target”polymerase inhibition of NRTIs. That is, the substitution of an R groupon NRTIs completely changed their activity towards polymeraseinhibition.

FIG. 93 shows that NRTIs, but not Kamuvudines, blocked mitochondrial DNAcopy number in primary human RPE cells in culture. NRTIs depletemitochondrial (mt) DNA by competing with endogenous nucleotides for DNApolymerase gamma; mtDNA depletion is thought to be largely responsiblefor many side effects associated with NRTI use. There were somedifferences between Kamuvudines in the rescue of mtDNA levels,indicating the need to test each modified compound individually. Thegraphs show the relative quantity of mtDNA in primary human RPE cellstreated with NRTIs (d4T, 3TC, or AZT) or modified NRTIs (Me-d4T,2Me-d4T, TM-3TC Me-AZT, or 2Me-AZT), relative to vehicle treatment(DMSO, a.k.a “No Tx”) at a concentration of 50 μM for all drugs. DNA wascollected after four days in cell culture with exposure to drug.Quantitative polymerase chain reaction was performed for mtDNA andnormalized to genomic DNA sequence. D4T, 3TC, and AZT exhibit mtDNAdepletion. Modified versions of d4T and AZT do not exhibit mtDNAdepletion. N=3-4/group, error bars are S.E.M. These data indicate thatNRTIs can cause mitochondrial toxicity, whereas most modified NRTIs donot do cause mitochondrial toxicity. As mitochondrial toxicity has beenblamed for myopathy, peripheral neuropathy, and hepatic steatosis withlactic acidosis observed in patients taking NRTIs, and the modifiedNRTIs do not cause mitochondrial toxicity, it is expected that themodified NRTIs are safer (i.e., decrease or eliminate motichondrialtoxicity) than the original NRTIs.

FIG. 94 shows the results of a L1 retrotransposition assay, in which anEGFP reporter is expressed only upon successful reverse transcriptionand integration of human LINE-1 retrotransposon. NRTIs, but notKamuvudines, blocked L1 retrotransposition, indicating that they do notblock RNA-dependent DNA polymerase activity. These findings pave way forthe rational re-design and improvement of nucleoside analogs as noveltherapeutics. The enhanced green fluorescent protein (EGFP) cell cultureL1retrotransposition assay was performed in HeLa cells. Cells weretransfected with a plasmid expressing an active L1 sequence tagged withan EGFP reporter in the presence of vehicle (RPS) or in the presence ofNRTIs (d4T, AZT, or 3TC) or methoxy-NRTIs (Me-d4T, 2Me-d4T, Me-AZT,2Me-AZT, 3Me-3TC, 2Et-d4T, 2Et-AZT, or 3Et-3TC). Transfected cells wereselected in puromycin. 7 days after transfection, cells that underwentretrotransposition (EGFP-positive) were assayed by flow cytometry. Cellswere gated based on background fluorescence of plasmid JM111, which hastwo point mutations in L1-ORF1 that abolish retrotransposition. Data arenormalized with RPS set to 1. These data indicate that NRTIs interferewith endogenous L1 activity whereas the modified NRTIs would notinterfere with this natural L1 activity of the cell. 3Et-3TC appears tohave some residual interference with natural L1 activity, whereas theother Kamuvudines (modified NRTIs) have no effect on L1 activity.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference,including the references set forth in the following list:

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One of ordinary skill in the art will recognize that additionalembodiments or implementations are possible without departing from theteachings of the present disclosure or the scope of the claims whichfollow. This detailed description, and particularly the specific detailsof the exemplary embodiments and implementations disclosed herein, isgiven primarily for clarity of understanding, and no unnecessarylimitations are to be understood therefrom, for modifications willbecome obvious to those skilled in the art upon reading this disclosureand may be made without departing from the spirit or scope of theclaimed invention.

1. A method of treating a condition selected from Parkinson's disease,Alzheimer's disease, and multiple sclerosis comprising administering toa subject in need thereof a compound of formula I

wherein R¹ is CH₃ or CH₂CH₃; and R² is H, CH₃, or CH₂CH₃, provided thatwhen R² is H, R¹ is not CH₃.
 2. The method of claim 1, wherein R² is CH₃or CH₂CH₃.
 3. A method of treating a condition selected from Parkinson'sdisease, Alzheimer's disease, and multiple sclerosis comprisingadministering to a subject in need thereof a compound having a structureselected from:

and pharmaceutically acceptable salts thereof.
 4. The method of claim 3,wherein the compound has a structure selected from:

and pharmaceutically acceptable salts thereof.
 5. The method of claim 3,wherein the compound has a structure selected from:

and pharmaceutically acceptable salts thereof.
 6. The method of claim 3,wherein the compound has a structure selected from:


7. The method of claim 3, wherein the compound has the followingstructure:


8. The method of claim 1, wherein the method comprises administering acomposition comprising the compound of formula I and a pharmaceuticallyacceptable carrier.
 9. The method of claim 1, wherein the condition isParkinson's disease.
 10. The method of claim 1, wherein the condition isAlzheimer's disease.
 11. The method of claim 1, wherein the condition ismultiple sclerosis.
 12. The method of claim 3, wherein the methodcomprises administering a composition comprising the compound and apharmaceutically acceptable carrier.
 13. The method of claim 3, whereinthe condition is Parkinson's disease.
 14. The method of claim 3, whereinthe condition is Alzheimer's disease.
 15. The method of claim 3, whereinthe condition is multiple sclerosis.